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• • A—HUMAN NECESSITIES
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  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
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  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
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  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• the invention relates to the field of protein biochemistry.
• the invention relates to the field of compositions and methods for producing proteins that include unnatural amino acids.
• amino acids can be modified by posttranslational modification of proteins, e.g., glycosylation, phosphorylation or oxidation, or in rarer instances, by the enzymatic modification of aminoacylated suppressor tRNAs, e.g., in the case of selenocysteine. Nonetheless, polypeptides, which are synthesized from only these 20 simple building blocks, carry out all of the complex processes of life. [06] Both site-directed and random mutagenesis, in which specific amino acids in a protein can be replaced with any of the other nineteen common amino acids, have become important tools for understanding the relationship between the structure and function of proteins. These methodologies have made possible the generation of proteins with enhanced properties, including stability, catalytic activity and binding specificity.
• the properties of proteins can be modified as compared to a protein composed of only amino acids from the 20 common amino acids, e.g., as in a naturally occurring protein.
• Several strategies have been employed to introduce unnatural amino acids into proteins. The first experiments involved the derivatization of amino acids with reactive side-chains such as Lys, Cys and Tyr, for example, the conversion of lysine to N 2 -acetyl- lysine.
• Chemical synthesis also provides a straightforward method to incorporate unnatural amino acids, but routine solid-phase peptide synthesis is generally limited to small peptides or proteins with less than 100 residues. With the recent development of enzymatic ligation and native chemical ligation of peptide fragments, it is possible to make larger proteins, but the method is not easily scaled. See, e.g., P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem, 69:923 (2000).
• a general in vitro biosynthetic method in which a suppressor tRNA chemically acylated with the desired unnatural amino acid is added to an in vitro extract capable of supporting protein biosynthesis has been used to site-specifically incorporate over 100 unnatural amino acids into a variety of proteins of virtually any size. See, e.g., V. W. Cornish, D. Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Enel.. 1995, 34:621 (1995); C.J. Noren, S.J. Anthony-Cahill, M.C. Griffith, P.G. Schultz, A general method for site-specific incorporation of unnatural amino acids into proteins, Science 244 182-188 (1989); and, J.D. Bain, C.G. Glabe, T.A. Dix, A.R.
• An auxotrophic strain in which the relevant metabolic pathway supplying the cell with a particular natural amino acid is switched off, is grown in minimal media containing limited concentrations of the natural amino acid, while transcription of the target gene is repressed. At the onset of a stationary growth phase, the natural amino acid is depleted and replaced with the unnatural amino acid analog. Induction of expression of the recombinant protein results in the accumulation of a protein containing the unnatural analog.
• an alternative strategy to inco ⁇ orate unnatural amino acids into proteins in vivo is to modify synthetases that have proofreading mechanisms. These synthetases cannot discriminate and therefore activate amino acids that are structurally similar to the cognate natural amino acids. This error is corrected at a separate site, which deacylates the mischarged amino acid from the tRNA to maintain the fidelity of protein translation. If the proofreading activity of the synthetase is disabled, structural analogs that are misactivated may escape the editing function and be inco ⁇ orated. This approach has been demonstrated recently with the valyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A. Nangle, T. L. Hendrickson, V.
• ValRS valyl-tRNA synthetase
• ValRS can misaminoacylate tRNAVal with Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids are subsequently hydrolyzed by the editing domain. After random mutagenesis of the Escherichia coli chromosome, a mutant Escherichia coli strain was selected that has a mutation in the editing site of ValRS. This edit-defective ValRS incorrectly charges tRNAVal with Cys.
• At least one major limitation of the methods described above is that all sites corresponding to a particular natural amino acid throughout the protein are replaced.
• the extent of inco ⁇ oration of the natural and unnatural amino acid may also vary - only in rare cases can quantitative substitution be achieved since it is difficult to completely deplete the cognate natural amino acid inside the cell.
• Another limitation is that these strategies make it difficult to study the mutant protein in living cells, because the multisite inco ⁇ oration of analogs often results in toxicity.
• this method is applicable in general only to close structural analogs of the common amino acids, again because substitutions must be tolerated at all sites in the genome.
• Solid-phase synthesis and semisynthetic methods have also allowed for the synthesis of a number of small proteins containing novel amino acids.
• a suppressor tRNA was prepared that recognized the stop codon UAG and was chemically aminoacylated with an unnatural amino acid.
• Conventional site- directed mutagenesis was used to introduce the stop codon TAG, at the site of interest in the protein gene. See, e.g., Sayers, J.R., Schmidt, W. Eckstein, F. 5', 3' Exonuclease in phosphorothioate-based olignoucleotide-directed mutagensis, Nucleic Acids Res, 791-802 (1988).
• a Xenopus oocyte was coinjected with two RNA species made in vitro: an mRNA encoding the target protein with a UAG stop codon at the amino acid position of interest and an amber suppressor tRNA aminoacylated with the desired unnatural amino acid.
• the translational machinery of the oocyte then inserts the unnatural amino acid at the position specified by UAG. This method has allowed in vivo structure-function studies of integral membrane proteins, which are generally not amenable to in vitro expression systems.
• Examples include the inco ⁇ oration of a fluorescent amino acid into tachykinin neurokinin-2 receptor to measure distances by fluorescence resonance energy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U. Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J. Biol. Chem., 271:19991 (1996); the inco ⁇ oration of biotinylated amino acids to identify surface-exposed residues in ion channels, see, e.g., J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem.
• the suppressor tRNA has to be chemically aminoacylated with the unnatural amino acid in vitro, and the acylated tRNA is consumed as a stoichiometric reagent during translation and cannot be regenerated.
• This limitation results in poor suppression efficiency and low protein yields, necessitating highly sensitive techniques to assay the mutant protein such as electrophysiological measurements.
• this method is only applicable to cells that can be microinjected.
• yeast amber suppressor tRNAPheCUA /phenylalanyl-tRNA synthetase pair was used in a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See, e.g., R. Furter, Protein Sci.. 7:419 (1998). Because yeast PheRS does not have high substrate specificity for p-F-Phe, the mutagenesis site was translated with only 64-15% p-F-Phe and the remainder as Phe and Lys even in the excess of p-F-Phe added to the growth media.
• the present invention provides a variety of methods for making and using translation systems that can inco ⁇ orate unnatural amino acids into proteins, as well as related compositions.
• Proteins comprising unnatural amino acids made by the translation system are also a feature of the invention. Both known and new unnatural amino acids can be inco ⁇ orated into proteins using the translation system of the invention.
• the invention further provides novel unnatural amino acids; various compositions including the unnatural amino acids, e.g., proteins and cells including unnatural amino acids; chemical and biosynthetic methods for producing unnnatural amino acids; and methods for producing and compositions comprising an autonomous twenty-one amino acid cell.
• the present invenion provides compositions comprising a translation system.
• the translation system comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS).
• O-tRNA orthogonal tRNA
• O-RS orthogonal aminoacyl tRNA synthetase
• the O-RS preferentially aminoacylates the O-tRNA with at least one unnatural amino acid in the translation system and the O-tRNA recognizes at least one selector codon.
• the translation system thus inserts the unnatural amino acid into a protein produced in the system, in response to an encoded selector codon.
• Typical translation systems include cells, such as bacterial cells (e.g., Escherichia coli), archeaebacterial cells, eukaryotic cells (e.g., yeast cells, mammalian cells, plant cells, insect cells), or the like.
• the translation system comprises an in vitro translation system, e.g., a translation extract including a cellular extract.
• Example O-tRNAs comprise a nucleic acid comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 1-3 and/or a complementary polynucleotide sequence thereof.
• example O-RS include polypeptides selected from the group consisting of: a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 35-66 and a polypeptide encoded by a nucleic acid comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 4-34 and a complementary polynucleotide sequence thereof.
• Examples of unnatural amino acids that can be used by the translation system include: an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a
• the unnatural amino acid can be an O-methyl-L-tyrosine, an L-3-(2- naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAc ⁇ -serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L- phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L- phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a -iodo- phenylalanine, a -bromophenylalanine, ap-amino-L-pheny
• the at least one unnatural amino acid is an O-methyl-L- tyrosine. In one specific example embodiment, the at least one unnatural amino acid is an L-3-(2-naphthyl)alanine. In another set of specific examples, the at least one unnatural amino acid is an amino-, isopropyl-, or O-allyl-containing phenylalanine analogue.
• Any of a variety of selector codons can be used in the present invention, including nonsense codons, rare codons, four (or more) base codons, or the like.
• the at least one selector codon is an amber codon.
• a variety of exemplar translation systems are provided herein, including e.g., an Escherichia coli cell comprising a mtRNA ⁇ and a mutant TyrRS (LWJ16), where the mutant TyrRS (LWJ16) preferentially aminoacylates the mtRNA ⁇ A with O-methyl-L- tyrosine in the cell and the cell uses the mtRNA ⁇ A to recognize an amber codon.
• an Escherichia coli cell comprising a mtRNA ⁇ A and an SS12-TyrRS is provided, where the SS12-TyrRS preferentially aminoacylates the mtRNAju A with L-3-
• the translation system herein provides the ability to synthesize proteins that comprise unnatural amino acids in usefully large quantities.
• proteins comprising at least one unnatural amino acid can be produced at a concentration of at least about 10, 50, 100 or more micrograms per liter, e.g., in a composition comprising a cell extract, a buffer, a pharmaceutically acceptable excipient, and/or the like.
• Another aspect of the present invention provides for the production of proteins that are homologous to any available protein, but comprising one or more unnatural amino acid homologue.
• therapeutic proteins can be made that comprise one or more unnatural amino acid and are homologous to one or more therapeutic protein.
• the protein is homologous to a therapeutic or other protein such as: a cytokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule, a steroid hormone receptor, a transcriptional activator, a transcriptional suppressor, erythropoietin (EPO), insulin, human growth hormone, epithelial Neutrophil Activating Peptide-78, GRO ⁇ /MGSA, GRO ⁇ , GRO ⁇ , MlP-l , MlP-l ⁇ , MCP-1, hepatocyte growth factor, insulin-like growth factor, leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, pleiotropin, SCF, c-kit ligand, VEGEF, G-CSF, LL-1, IL-2, IL-8, IGF-I, IGF- ⁇ , F
• the protein is homologous to a therapeutic or other protein such as: an Alpha-1 antitrypsin, an Angiostatin, an Antihemolytic factor, an antibody, an Apolipoprotein, an Apoprotein, an Atrial natriuretic factor, an Atrial natriuretic polypeptide, an Atrial peptide, a C-X-C chemokine, T39765, NAP-2, ENA-78, a Gro-a, a Gro-b, a Gro-c, an IP-10, a GCP-2, an NAP-4, an SDF-1, a PF4, a MIG, a Calcitonin, a c-kit ligand, a cytokine, a CC chemokine, a Monocyte chemoattractant protein- 1, a Monocyte chemoattractant protein-2, a Monocyte chemoattractant protein-3, a Monocyte inflammatory protein- 1 alpha, a Monocyte inflammatory protein- 1 alpha,
• Homology to the polypeptide can be inferred by performing a sequence alignment, e.g., using BLASTN or BLASTP, e.g., set to default parameters.
• the protein is at least about 50%, at least about 75%, at least about 80%, at least about 90% or at least about 95% identical to a known therapeutic protein (e.g., a protein present in Genebank or other available databases).
• the therapeutic protein is erythropoeitin (EPO).
• the protein of interest can contain 1, 2, 3, 4, 5, 6, 7, 6, 9, 10, 11, 12, 13, 14, 15 or more unnatural amino acids.
• the unnatural amino acids can be the same or different, e.g., there can be 1, 2, 3, 4, 5, 6, 7, 6, 9, 10, 11, 12, 13, 14, 15 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 6, 9, 10, 11, 12, 13, 14, 15 or more different unnatural amino acids.
• the protein is DHFR, and the at least one unnatural amino acid is selected from the group consisting of O-methyl-L- tyrosine and L-3-(2-naphthyl)alanine.
• the present invention also provides methods for producing at least one protein in a translation system such that the at least one protein comprises at least one unnatural amino acid.
• the translation system is provided with at least one nucleic acid comprising at least one selector codon, wherein the nucleic acid encodes the at least one protein.
• the translation system is also provided with an orthogonal tRNA (O-tRNA), that functions in the translation system and recognizes the at least one selector codon and an orthogonal aminoacyl tRNA synthetase (O-RS), that preferentially aminoacylates the O-tRNA with the at least one unnatural amino acid in the translation system.
• O-tRNA orthogonal tRNA
• O-RS orthogonal aminoacyl tRNA synthetase
• the translation system is also provided with the at least one unnatural amino, thereby producing, in the translation system, the at least one protein comprising the at least one unnatural amino acid.
• compositions can be embodied in the methods, e.g., types of translation systems (e.g., cells, cell extracts, etc.), types of proteins produced in the translation systems (e.g., EPO homologues and the other proteins noted herein) specific mutant proteins, specific unnatural amino acids, and the like.
• types of translation systems e.g., cells, cell extracts, etc.
• proteins produced in the translation systems e.g., EPO homologues and the other proteins noted herein
• specific mutant proteins e.g., specific unnatural amino acids, and the like.
• the protein(s) comprising unnatural amino acids that are produced are processed and modified in a cell-dependent manner. This provides for the production of proteins that are stably folded, glycosylated, or otherwise modified by the cell.
• the unnatural amino acid is optionally provided exogenously to the translation system. Alternately, e.g., where the translation system is a cell, the unnatural amino acid can be biosynthesized by the translation system.
• the invention provides methods for producing in an Escherichia coli cell at least one protein comprising at least one O-methyl- L-tyrosine.
• the method includes providing the translation system with at least one nucleic acid comprising an amber codon, wherein the nucleic acid encodes the at least one protein; providing the translation system with a mtRNA ⁇ , wherein the mtRNA ⁇ functions in the cell and wherein the mtRNAju A recognizes the amber codon; providing the translation system with a mutant TyrRS (LWJ16), wherein the mutant TyrRS (LWJ16) aminoacylates the mtRNA ⁇ u ⁇ with the O-methyl-L-tyrosine in the cell; and, providing the cell with the
• O-methyl-L-tyrosine thereby producing in the cell at least one protein comprising the O- methyl-L-tyrosine.
• the invention provides a method for producing in an Escherichia coli cell at least one protein comprising at least one L-3-(2- naphthyl)alanine.
• the method includes: providing the translation system with at least one nucleic acid comprising an amber codon, wherein the nucleic acid encodes the at least one protein; providing the cell with a mtRNA ⁇ , wherein the mtRNA ⁇ functions in the cell and wherein the mtRNA ⁇ recognizes the amber codon; providing the cell with an SS12-TyrRS, wherein the SS12-TyrRS aminoacylates the mtRNAj ⁇ with the L-3-(2-naphthyl)alanine in the cell; and, providing the cell with the L-3-(2-naphthyl)alanine, thereby producing in the cell at least one protein comprising the L-3-(2-naphthyl)alanine.
• the present invention provides unnatural amino acids, e.g., meta substituted phenylalanine analogues, such as 3-acetyl-phenylalanine and 3-methoxy phenylalanine; tyrosine analogues, such as 4-allyl tyrosine; glycosylated amino acids, and the like.
• unnatural amino acids e.g., meta substituted phenylalanine analogues, such as 3-acetyl-phenylalanine and 3-methoxy phenylalanine
• tyrosine analogues such as 4-allyl tyrosine
• glycosylated amino acids and the like.
• compositions comprising unnatural amino acids e.g., proteins and cells comprising the unnatural amino acids of the invention, are also provided.
• compositions comprising an unnatural amino acid and an orthogonal tRNA, e.g., covalently bonded are provided.
• compositions comprising unnatural amino acids and an orthogonal aminoacyl tRNA synthetase, e.g., hydrogen bonded, are also provided.
• the present invention provides methods of synthesizing amino acids.
• 4-allyl-L-tyrosine is typically synthesized by reacting a protected tyrosine with allyl bromide, e.g., in the presence of sodium hydride and DMF, and deprotecting to yield 4-allyl-L-tyrosine.
• an NBoc or Fmoc protected tyrosine is used, e.g., with an acidic deprotection, e.g., in the presence of hydrochloric acid and dioxane.
• Meta-substituted phenylalanine analogues are typically synthesized by condensing diethylacetamidomalonate and a meta-substituted benzyl bromide. The product of the condensation is then typically hydrolyzed to yield the meta-substituted phenylalanine analogue, e.g., a keto, acetyl, or methoxy substituted phenylalanine such as 3-methoxy- phenylalanine or 3-acetyl-phenylalanine.
• a keto, acetyl, or methoxy substituted phenylalanine such as 3-methoxy- phenylalanine or 3-acetyl-phenylalanine.
• the desired meta substituted benzyl bromide is optionally synthesized by reacting N-bromosuccinimide (NBS) with 3- methylacetophenone to produce a brominated product, and crystallizing the brominated product in a hexane solution.
• NBS N-bromosuccinimide
• the crystallization yields a monobromide product as opposed to a mixture of a monobromide and a dibromide.
• the present invention provides biosynthetic methods for producing unnatural amino acids.
• glycosylated amino acids are optionally synthesized in vivo, e.g., by transforming a cell with a plasmid comprising a gene for an N-acetyl-galactosaminidase, a transglycosylase, or a serine-glycosylhydrolase. The cell then produces the desired glycosylated amino acid, e.g. from cellular resources.
• p-aminophenylalanine is synthesized, e.g., in vivo, by enzymatically converting chorismate to 4-amino-4-deoxychorismic acid; which is enzymatically converted to 4- amino-4-deoxyprephenic acid; and enzymatically converting the 4-amino-4- deoxyprephenic acid to p-aminophenyl-pyruvic acid, which is enzymatically converted to
• the enzymatic conversions are typically performed using a 4- amino-4-deoxychorismate synthase, e.g., PapA, a chorismate mutase, e.g., Pap B, and a prephenate dehydrogenase, e.g., PapC, respectively.
• the final step is typically performed by contacting the p-aminophenyl-pyruvic acid with an aminotransferease, e.g., .a nonspecific tyrosine aminotransferase, e.g., derived from E coli.
• Aminotransfereases of use in the present invention include, but are not limited to, tyrB, aspS, or ilv ⁇ .
• the above steps are performed in vivo, e.g., by transforming a cell with a plasmid comprising the genes which encode the enzymes used for the synthesis.
• the present invention provides a method of producing p- aminophenylalanine in an Escherichia coli cell. The method typically comprises transforming the cell with a plasmid comprising papA, papB, and papC, wherein the cell comprises chorismate and an aminotransferase.
• pap A, papB, and papC results in a synthase, a mutase, and a dehydrogenase, wherein these enzymes together with the aminotransferase produce p-phenylalanine from chorismate.
• the present invention provides an autonomous twenty-one (or more) amino acid cell.
• the cell e.g., a bacterial cell
• O-tRNA orthogonal tRNA
• O-RS orthogonal aminoacyl tRNA synthetase
• the O-RS preferentially aminoacylates the O-tRNA with the unnatural amino acid and the O-tRNA inco ⁇ orates the unnatural amino acid into a protein in response to a selector codon, e.g., a nonsense codon such as TAG, a four base codon, or an amber codon.
• a selector codon e.g., a nonsense codon such as TAG, a four base codon, or an amber codon.
• the cell can comprise more than one unnatural amino acid, e.g.
• unnatural amino acids optionally with more than one orthogonal tRNA (e.g., one per unnatural amino acid to provide for site-specific inco ⁇ oration of each unnatural amino acid in a protein, or more, or less, to tune the specificity of unnatural amino acid inco ⁇ oration) and/or more than one orthogonal aminoacyl tRNA synthetase (O-RS) (e.g., one per orthogonal tRNA, or more or less to tune the specificity of unnatural amino acid inco ⁇ oration).
• orthogonal tRNA e.g., one per unnatural amino acid to provide for site-specific inco ⁇ oration of each unnatural amino acid in a protein, or more, or less, to tune the specificity of unnatural amino acid inco ⁇ oration
• OF-RS orthogonal aminoacyl tRNA synthetase
• the biosynthetic pathway systems produce a natural cellular amount of the unnatural amino acid, e.g., the cell produces the unnatural amino acid in an amount sufficient for protein biosynthesis, which amount does not substantially alter the concentration of natural amino acids or substantially exhaust cellular resources in the production of the unnatural amino acids.
• the autonomous cell is engineered to produce p-aminophenylalanine from chorismate as described above.
• the cell is engineered to produce the desired enzymes as described above, e.g., a synthase, a dehydrogenase, and a mutase derived from Streptomyces Venezuelae or Streptomyces pristinaespiralis and a aminotransferase derived from E.coli.
• the cells of the invention are optionally transformed with a plasmid, e.g., low copy pSClOl derived plasmid, comprising papA, papB, and papC, wherein the plasmid further comprises an lpp promoter and a lac promoter.
• the plasmid further comprises one or more ribosome binding sites.
• the present invention provides a cell comprising one or more systems for producing at least twenty one amino acids and specifically inco ⁇ orating one or more of the amino acids into one or more proteins within the cell, wherein at least one of the inco ⁇ orated amino acids comprises an unnatural amino acid.
• the present invention provides a method of identifying an advantage provided by an unnatural amino acid which has been inco ⁇ orated into one or more proteins of a cell.
• the method typically comprises providing a library of cells, each of which cells comprises a randomized plasmid, e.g., derived from an E. coli genome.
• a randomized plasmid e.g., derived from an E. coli genome.
• One or more of the randomized plasmids typically confers on the cells an ability to inco ⁇ orate an unnatural amino acid into a protein.
• the library of cells is then screened to identify cells with enhanced growth, e.g., as compared to a native E. coli cell, thereby identifying an advantage provided by the unnatural amino acid.
• a second screen is used to further verify that any advantage identified is due to the unnatural amino acid.
• kits are an additional feature of the invention.
• the kits can include one or more translation system as noted above (e.g., a cell, a 21 or more amino acid cell, etc.), one or more unnatural amino acid, e.g., with appropriate packaging material, containers for holding the components of the kit, instructional materials for practicing the methods herein and/or the like.
• products of the translation systems e.g., proteins such as ⁇ PO analogues comprising unnatural amino acids
• kit form e.g., with containers for holding the components of the kit, instructional materials for practicing the methods herein and/or the like.
• Figure 1 is a sterioview of the amino acid residues in the active site of TyrRS (modified from P. Brick, T. N. Bhat, D. M. Blow, J. Mol. Biol. 208, 83-98 (1988)). Residues from B. stearothermophilus TyrRS are shown in the figure.
• Figure 2 illustrates accumulation of E. coli DHFR protein, both wild-type (wt) and mutant under different conditions. Expression conditions are notated at the top of each lane. The left lane is molecular weight marker.
• Figure 2A is a silver-stained SDS-PAGE gel of purified DHFR.
• Figure 2B is a Western blot of the gel in Figure 2A.
• Figure 3 is a tandem mass spectrum of an NH2 terminal peptide of DHFR, MIY*MIAALAVDR. The partial sequence Y*MIAALAVDR of the peptide containing the O-methyl-L-tyrosine residue (Y*) can be read from the annotated b or y ion series.
• Figure 4 illustrates accumulation of mouse DHFR protein, both wild-type (wt) and mutant, under different conditions. Expression conditions are notated at the top of each lane. The left lane is molecular weight marker.
• Figure 4A is a silver-stained SDS-PAGE gel of purified DHFR.
• Figure 4B is a Western blot of the gel in Figure 4A.
• Figure 5 is a tandem mass spectrum of the tryptic peptide LLPEX*TGVLSEVQEEK (X* represents L-3-(2-naphthyl)-alanine). The sequence can be read from the annotated b or y ion series; even so, b7 and yl3 are not observed. The base peak 821.7 (100%) assigned to the doubly charged yl4 ion is truncated for clarity.
• Figure 6 Panels A-D, illustrate features of the amplifiable fluorescence reporter system.
• Figure 6A is plasmid pREP.
• T7 RNA polymerase transcription is controlled by the ara promoter; protein expression depends on suppression of amber codons at varying locations within the gene. GFPuv expression is controlled by T7 RNA polymerase.
• Plasmid pREP is compatible for use with a ColEl plasmid expressing an orthogonal synthetase/tRNA pair.
• Figure 6B illustrates composition and fluorescence enhancement of T7 RNA polymerase gene constructs within pREP(l-12). The construct number is indicated to the left of each. Fluorescence enhancements, indicated to the right of each construct, are calculated as the cell concentration-corrected ratio of fluorescence, as measured fluorimetrically, of cells containing pREP(l-12) and pQ or pQD. The positions of amber mutations within the gene are indicated above each construct.
• Figure 6C illustrates cytometric analysis of cells containing pREP(10) and either pQD (top) or pQ
• Figure 6D illustrates fluorimetric analyses of cells containing pREP(10) and expressing various E. coli suppressor tRNAs. 'None' indicates that the cells contain no suppressor tRNA.
• Figure 7A illustrates plasmid pREP/YC-JYCUA. Plasmid pREP/YC-JYCUA is compatible for use with plasmid pBK and variants.
• Figure 7B illustrates structures of unnatural amino acids used as targets for the evolution of M. jannaschii TyrRS.
• Figure 7C illustrates a strategy for a evolution of an aminoacyl-tRNA synthetase using plasmid pREP/YC-JYCUA. Fluorescent and non- fluorescent cells are shown in black and grey, respectively.
• Figure 8 Panels A-D, illustrates the activity of the dominant synthetase variant from each successful evolution experiment.
• Figure 8A is a photograph illustrating long- wavelength ultraviolet illumination of cells containing pREP/YC-JYCUA and the indicated synthetase variant, grown in either the presence (+) or absence (-) of the corresponding unnatural amino acid.
• Figure 8B illustrates a fluorimetric analysis of cells containing pREP/YC-JYCUA and the indicated synthetase variant, grown in either the presence (left) or absence (right) of the corresponding unnatural amino acid.
• Figure 8C is a table that illustrates a Cm ICso analysis of cells containing pREP/YC-JYCUA and the indicated synthetase variant, grown in either the presence or absence of the corresponding unnatural amino acid.
• Figure 8D illustrates a protein expression analysis from cells containing pBAD/JYAMB-4TAG and the indicated synthetase variant, grown in either the presence (+) or absence (-) of the corresponding unnatural amino acid.
• Figure 9 illustrates activity comparisons of OAY-RS variants derived using a negative FACS-based screen [OAY-RS(1,3,5)] or negative barnase-based selection [OAY- RS(B)].
• FIG. 10 is an autoradiograph of a western blot demonstrating expression of m- MeO-Phe- and m-Acetyl-Phe- inco ⁇ orated DHFR.
• Figure 11 illustrates the fluorescence emission spectra of fluorescein hydrazide labelled protein.
• Figure 12 illustrates the unnatural amino acids para-azido-phelylalanine and para- benzoyl-phenylalanine.
• Figure 13 illustrates a chemical scheme for the synthesis of an allyl-substituted phenylalanine.
• Figure 14 illustrates a chemical scheme for the synthesis of meta-substituted phenylalanines.
• Figure 15 illustrates the biosynthesis of/?-aminophenylalanine.
• Panel A illustrates a plasmid used for the biosynthesis of -aminophenylalanine
• Panel B illustrates a biosynthetic scheme for the production of p-aminophenylalanine from chorismate, e.g., using the plasmid of Panel A.
• Figure 16 illustrates a variety of unnatural amino acids.
• Figure 17 illustrates a variety of unnatural amino acids.
• Figure 18 illustrates a variety of unnatural amino acids.
• Figure 19 illustrates additional amino acids, natural and unnatural for inco ⁇ oration into proteins via in vivo suppression.
• Figure 20 provides a biosynthetic scheme for production of dopa.
• Figure 21 illustrates a method for determining evolutionary advantages in a cell due to the ability to specifically inco ⁇ orate twenty-one amino acids.
• Figure 22 illustrates a method for site-specific inco ⁇ oration of unnatural amino acids.
• FIG. 23 illustrates the synthesis of various glutamine analogs.
• Figure 24 illustrates the synthesis of a gamma substituted glutamine analog.
• Figure 25 illustrates the synthesis of a cyclic glutamine derivative.
• Figure 26 illustrates a variety of tyrosine analogs.
• Figure 27 illustrates a synthetic scheme for the production of tyrosine analogs.
• Figure 28 illustrates a biosynthetic scheme for producing glycosylated amino acids.
• Figure 29 illustrates a variety of unnatural amino acids, e.g., as used in a cellular uptake study. Any or all of the above figures are schematic in nature.
• the present invention provides compositions and methods for augmenting the protein biosynthetic machinery of a cell to accommodate additional genetically encoded amino acids using orthogonal tRNA/aminoacyl tRNA synthetase (O-tRNA/O-RS) pairs.
• the compositions and methods described here can be used with unnatural amino acids, e.g., providing novel spectroscopic, chemical or structural properties to proteins using any of a wide array of side chains.
• the invention is applicable to both prokaryotic (e.g., Eubacteria, Archeaebacteria) and eukaryotic (e.g., yeast, mammalian, plant, or insect) cells.
• compositions and methods are useful for the site specific inco ⁇ oration of unnatural amino acids via selector codons, e.g., stop codons, four base codons, and the like.
• the invention also provides proteins, including unnatural amino acids, produced using the compositions or made by the methods of the invention.
• the ability to introduce unnatural amino acids into proteins directly in living cells provides new tools for studies of protein and cellular function and can lead to the generation of proteins with enhanced properties useful for, e.g., therapeutics.
• Homologous Proteins and/or protein sequences are "homologous" when they are derived, naturally or artificially, from a common ancestral protein or protein sequence.
• nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence.
• any naturally occurring nucleic acid can be modified by any available mutagenesis method to include one or more selector codon. When expressed, this mutagenized nucleic acid encodes a polypeptide comprising one or more unnatural amino acid.
• the mutation process can, of course, additionally alter one or more standard codon, thereby changing one or more standard amino acid in the resulting mutant protein as well.
• Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof).
• the precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology.
• Higher levels of sequence similarity e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology.
• Methods for determining sequence similarity percentages e.g., BLASTP and BLASTN using default parameters are described herein and are generally available.
• Orthogonal refers to a molecule (e.g., an orthogonal tRNA (O-tRNA) and or an orthogonal aminoacyl tRNA synthetase (O-RS)) that is used with reduced efficiency by a system of interest (e.g., a translational system, e.g., a cell).
• Orthogonal refers to the inability or reduced efficiency, e.g., less than 20 % efficient, less than 10 % efficient, less than 5 % efficient, or e.g., less than 1% efficient, of an orthogonal tRNA and/or orthogonal RS to function in the translation system of interest.
• an orthogonal tRNA in a translation system of interest aminoacylates any endogenous RS of a translation system of interest with reduced or even zero efficiency, when compared to aminoacylation of an endogenous tRNA by the endogenous RS.
• Preferentially aminoacylates refers to an efficiency of, e.g., about 70 % efficient, about 75 % efficient, about 85% efficient, about 90% efficient, about 95 % efficient, or about 99% or more efficient, at which an O-RS aminoacylates an O-tRNA with an unnatural amino acid compared to a naturally occurring tRNA or starting material used to generate the O-tRNA.
• the unnatural amino acid is then inco ⁇ orated into a growing polypeptide chain with high fidelity, e.g., at greater than about 75% efficiency for a given selector codon, at greater than about 80% efficiency for a given selector codon, at greater than about 90% efficiency for a given selector codon, at greater than about 95% efficiency for a given selector codon, or at greater than about 99% or more efficiency for a given selector codon.
• Selector codon refers to codons recognized by the O- tRNA in the translation process and not recognized by an endogenous tRNA.
• the O- tRNA anticodon loop recognizes the selector codon on the mRNA and inco ⁇ orates its amino acid, e.g., an unnatural amino acid, at this site in the polypeptide.
• Selector codons can include, e.g., nonsense codons, such as, stop codons, e.g., amber, ochre, and opal codons; four or more base codons; codons derived from natural or unnatural base pairs and the like.
• a selector codon can also include one of the natural three base codons, wherein the endogenous system does not use said natural three base codon, e.g., a system that is lacking a tRNA that recognizes the natural three base codon or a system wherein the natural three base codon is a rare codon.
• a suppressor tRNA is a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system.
• a suppressor tRNA can read through, e.g., a stop codon, a four base codon, or a rare codon.
• Translation system refers to the components necessary to inco ⁇ orate a naturally occurring amino acid into a growing polypeptide chain (protein).
• Components of a translation system can include, e.g., ribosomes, tRNAs, synthetases, mRNA and the like.
• the components of the present invention can be added to a translation system, in vivo or in vitro.
• a translation system can be a cell, either prokaryotic, e.g., an E. coli cell, or eukaryotic, e.g., a yeast, mammalian, plant, or insect cell.
• Unnatural amino acid refers to any amino acid, modified amino acid, and/or amino acid analogue that is not one of the 20 naturally occurring amino acids or seleno cysteine.
• Proteins are at the crossroads of virtually every biological process, from photosynthesis and vision to signal transduction and the immune response. These complex functions result from a polyamide based polymer consisting of twenty relatively simple building blocks arranged in a defined primary sequence.
• the present invention includes methods and composition for use in the site-specific inco ⁇ oration of unnatural amino acids directly into proteins in vivo. Importantly, the unnatural amino acid is added to the genetic repertoire, rather than substituting for one of the common 20 amino acids.
• the present invention e.g., (i) allows the site-selective or random insertion of one or more unnatural amino acids at any desired position of any protein, (ii) is applicable to both prokaryotic and eukaryotic cells, (iii) enables in vivo studies of mutant proteins in addition to the generation of large quantities of purified mutant proteins, and (iv) is adaptable to inco ⁇ orate any of a large variety of non-natural amino acids into proteins in vivo.
• the invention provides compositions and methods useful for in vivo site specific inco ⁇ oration of unnatural amino acids.
• the invention provides translation systems, e.g., cells, that include an orthogonal tRNA (O- tRNA), an orthogonal aminoacyl tRNA synthetase (O-RS), and an unnatural amino acid, where the O-RS aminoacylates the O-tRNA with the unnatural amino acid, and the cell uses the components to inco ⁇ orate the unnatural amino acid into a growing polypeptide chain.
• O- tRNA orthogonal tRNA
• OF-RS orthogonal aminoacyl tRNA synthetase
• the invention further provides methods for in vivo site-specific inco ⁇ oration of unnatural amino acids using the translation systems of the invention.
• the invention also provides proteins produced by the methods of the invention.
• the claimed proteins include unnatural amino acids.
• compositions and methods of the invention utilize an orthogonal tRNA (O- tRNA) aminoacyl tRNA synthetase (O-RS) pair.
• O- tRNA orthogonal tRNA
• O-RS aminoacyl tRNA synthetase
• a wide range of pairs can be used with the following properties: the O-tRNA is preferentially aminoacylated with an unnatural amino acid by the O-RS.
• the orthogonal pair functions in the translation system of interest, e.g., the translation system uses the unnatural amino acid- aminoacylated O-tRNA to inco ⁇ orate the unnatural amino acid into a polypeptide chain.
• the O-tRNA recognizes a selector codon, e.g., a stop codon, in the mRNA coding for the protein.
• the O-tRNA is derived from a Tyr-tRNA from a Methanococcus jannaschii cell.
• the O-tRNA is that referred to herein as mtRNA u A •
• the O-tRNA includes a nucleic acid polynucleotide sequence selected from the group that includes SEQ ID NO: 1-3 or a complementary polynucleotide sequence thereof.
• the O-RS is derived from TyrRS from a Methanococcus jannaschii cell.
• the O-RS is referred to herein as mutant TyrRS (LWJ16) or SS12-TyrRS.
• the O-RS includes a polypeptide selected from the group consisting of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 35-66 and a polypeptide encoded by a nucleic acid comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 4-34 or a complementary polynucleotide sequence thereof.
• the invention includes an Escherichia coli cell comprising a mtRNAju A and a mutant TyrRS (LWJ16), wherein the mutant TyrRS
• the invention includes an Escherichia coli cell comprising a mtRNA ⁇ A and an SS12-TyrRS, wherein the SS12-TyrRS preferentially aminoacylates the mtRNA£u A with L-3-(2-naphthyl)alanine in the cell and the cell uses the mtRNA ⁇ u ⁇ to recognize an amber codon.
• An orthogonal pair is composed of an O-tRNA, e.g., a suppressor tRNA, a frameshift tRNA, or the like, and an O-RS.
• the O-tRNA is not acylated by endogenous synthetases and is capable of decoding a selector codon, as described above.
• the O-RS recognizes the O-tRNA, e.g., with an extended anticodon loop, and preferentially aminoacylates the O-tRNA with an unnatural amino acid.
• the development of multiple orthogonal tRNA/synthetase pairs can allow the simultaneous inco ⁇ oration of multiple unnatural amino acids using different codons.
• the O-tRNA and the O-RS can be naturally occurring or can be derived by mutation of a naturally occurring tRNA and/or RS from a variety of organisms, which are described under sources and hosts.
• the O-tRNA and O-RS are derived from at least one organism.
• the O-tRNA is derived from a naturally occurring or mutated naturally occurring tRNA from a first organism and the O-RS is derived from naturally occurring or mutated naturally occurring RS from a second organism.
• Patent application Serial No. titled “Methods and Compositions for the production of orthogonal tRNA-tRNA synthetase pairs," (Attorney docket number 54- 000130US by Schultz et al) filed concurrently with the instant application, the disclosure of which is inco ⁇ orated in its entirety.
• these methods solve the problems discussed in the background section for the other strategies that were attempted to generate orthogonal tRNA/RS pairs. Specifically, these methods include: (a) generating a library of tRNAs derived from at least one tRNA from a first organism; (b) negatively selecting the library for tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of a RS from the first organism, thereby providing a pool of tRNAs; (c) selecting the pool of tRNAs for members that are aminoacylated by an introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA.
• RS aminoacyl-tRNA synthetase
• the at least one recombinant O-tRNA recognizes a selector codon and is not efficiency recognized by the RS from the second organism and is preferentially aminoacylated by the O-RS.
• the method also includes: (d) generating a library of mutant RSs derived from at least one aminoacyl-tRNA synthetase (RS) from a third organism; (e) selecting the library of RSs for members that preferentially aminoacylate the at least one recombinant O-tRNA in the presence of an unnatural amino acid and a natural amino acid, thereby providing a pool of active RSs; and, (f) negatively selecting the pool for active RSs that preferentially aminoacylate the at least one recombinant O-tRNA in the absence of the unnatural amino acid, thereby providing the at least one specific O-tRNA/O-RS pair, where the at least one specific O-tRNA O-RS pair comprises at least one recombinant O-RS that is specific for the unnatural amino acid and
• One strategy for generating an orthogonal pair involves generating mutant libraries from wihc to screen and/or select an O-tRNA or O-RS.
• a second strategy for generating an orthogonal tRNA/synthetase pair involves importing a heterologous tRNA/synthetase pair, e.g., importing a pair from another, e.g., source organism into the host cell.
• the properties of the heterologous synthetase candidate include, e.g., that it does not charge any host cell tRNA, and the properties of the heterologous tRNA candidate include, e.g., that it is not acylated by any host cell synthetase.
• the heterologous tRNA derived from the heterologous tRNA is orthogonal to all host cell synthetases.
• O-RS orthogonal aminoacyl tRNA synthetases
• the selection methods of the present invention are: (i) sensitive, as the activity of desired synthetases from the initial rounds can be low and the population small; (ii) "tunable", since it is desirable to vary the selection stringency at different selection rounds; and, (iii) general, so that it can be used for different unnatural amino acids.
• Methods to generate an orthogonal aminoacyl tRNA synthetase include mutating the synthetase, e.g., at the active site in the synthetase, at the editing mechanism site in the synthetase, at different sites by combining different domains of synthetases, or the like, and applying a selection process.
• a strategy is used, which is based on the combination of a positive selection followed by a negative selection.
• the positive selection suppression of the selector codon introduced at a nonessential position(s) of a positive marker allows cells to survive under positive selection pressure.
• survivors thus encode active synthetases charging the orthogonal suppressor tRNA with either a natural or unnatural amino acid.
• the negative selection suppression of a selector codon introduced at a nonessential position(s) of a negative marker removes synthetases with natural amino acid specificities.
• Survivors of the negative and positive selection encode synthetases that aminoacylate (charge) the orthogonal suppressor tRNA with unnatural amino acids only.
• mutant RSs can then be subjected to further mutagenesis, e.g., DNA shuffling or other recursive mutagenesis methods.
• the library of mutant RSs can be generated using various mutagenesis techniques known in the art.
• the mutant RSs can be generated by site-specific mutations, random point mutations, homologous recombinantion, chimeric construction or the like.
• the positive selection step can include, e.g., introducing a positive selection marker, e.g., an antibiotic resistance gene, or the like, and the library of mutant RSs into a plurality of cells, wherein the positive selection marker comprises at least one selector codon, e.g., an amber codon; growing the plurality of cells in the presence of a selection agent; selecting cells that survive in the presence of the selection agent by suppressing the at least one selector codon in the positive selection marker, thereby providing a subset of positively selected cells that contains the pool of active mutant RSs.
• the selection agent concentration can be varied.
• the negative selection can include, e.g., introducing a negative selection marker with the pool of active mutant RSs from the positive selection into a plurality of cells of a second organism, wherein the negative selection marker is an antibiotic resistance gene, e.g., a chloramphenicol acetyltransferase (CAT) gene, comprising at least one selector codon; and, selecting cells that survive in a 1st media supplemented with the unnatural amino acid and a selection agent, but fail to survive in a 2nd media not supplemented with the unnatural amino acid and the selection agent, thereby providing surviving cells with the at least one recombinant O-RS.
• the concentration of the selection agent is varied.
• the positive selection can be based on suppression of a selector codon in a positive selection marker, e.g., a chloramphenicol acetyltransferase (CAT) gene comprising a selector codon, e.g., an amber stop codon, in the CAT gene, so that chloramphenicol can be applied as the positive selection pressure.
• a positive selection marker e.g., a chloramphenicol acetyltransferase (CAT) gene comprising a selector codon, e.g., an amber stop codon, in the CAT gene, so that chloramphenicol can be applied as the positive selection pressure.
• the CAT gene can be used as both a positive marker and negative marker as describe herein in the presence and absence of unnatural amino acid.
• the CAT gene comprising a selector codon is used for the positive selection and a negative selection marker, e.g., a toxic marker, such as a barnase gene comprising at least one or more selector codons, is used for the negative selection.
• a negative selection marker e.g., a toxic marker, such as a barnase gene comprising at least one or more selector codons
• the positive selection can also be based on suppression of a selector codon at a nonessential position in the ⁇ -lactamase gene, rendering cells ampicillin resistant; and a negative selection using the ribonuclease barnase as the negative marker is used.
• CAT localizes in the cytoplasm; moreover, ampicillin is bacteriocidal, while chloramphenicol is bacteriostatic.
• the recombinant O-RS can be further mutated and selected.
• the methods for producing at least one recombinant orthogonal aminoacyl-tRNA synthetase can further comprise: (d) isolating the at least one recombinant O-RS; (e) generating a second set of mutated O-RS derived from the at least one recombinant O- RS; and, (f) repeating steps (b) and (c) until a mutated O-RS is obtained that comprises an ability to preferentially aminoacylate the O-tRNA.
• steps (d)-(f) are repeated, e.g., at least about two times.
• the second set of mutated O-RS can be generated by mutagenesis, e.g., random mutagenesis, site-specific mutagenesis, recombination or a combination thereof.
• orthogonal tRNA recombinant orthogonal tRNA
• Methods of producing a recombinant O-tRNA include: (a) generating a library of mutant tRNAs derived from at least one tRNA, e.g., a suppressor tRNA, from a first organism; (b) negatively selecting the library for mutant tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of a RS from the first organism, thereby providing a pool of mutant tRNAs; and, (c) selecting the pool of mutant tRNAs for members that are aminoacylated by an introduced orthogonal RS (O- RS), thereby providing at least one recombinant O-tRNA; wherein the at least one recombinant O-tRNA recognizes a selector codon and is not efficiency recognized by the RS from the second organism and is preferentially aminoacylated by the O-RS.
• RS aminoacyl-tRNA synthetase
• the recombinant O-tRNA possesses an improvement of orthogonality.
• the methods include a combination of negative and positive selections with a mutant suppressor tRNA library in the absence and presence of the cognate synthetase, respectively.
• a selector codon(s) is introduced in a marker gene, e.g., a toxic gene, such as barnase, at a nonessential position.
• a member of the mutated tRNA library e.g., derived from Methanococcus jannaschii
• endogenous host e.g., Escherichia coli synthetases
• the selector codon e.g., an amber codon
• the toxic gene product produced leads to cell death.
• Cells harboring orthogonal tRNAs or non-functional tRNAs survive.
• Survivors are then subjected to a positive selection in which a selector codon, e.g., an amber codon, is placed in a positive marker gene, e.g., a drug resistance gene, such a ⁇ -lactamase gene.
• a positive marker gene e.g., a drug resistance gene, such a ⁇ -lactamase gene.
• These cells also contain an expression vector with a cognate RS.
• a selection agent e.g., ampicillin.
• tRNAs are then selected for their ability to be aminoacylated by the coexpressed cognate synthetase and to insert an amino acid in response to this selector codon.
• Cells harboring non-functional tRNAs, or tRNAs that cannot be recognized by the synthetase of interest are sensitive to the antibiotic.
• tRNAs that: (i) are not substrates for endogenous host, e.g., Escherichia coli, synthetases; (ii) can be aminoacylated by the synthetase of interest; and (iii) are functional in translation survive both selections.
• Libraries of mutated tRNA are constructed. Mutations can be introduced at a specific position(s), e.g., at a nonconservative position(s), or at a conservative position, at a randomized position(s), or a combination of both in a desired loop of a tRNA, e.g., an anticodon loop, (D arm, V loop, T ⁇ C arm) or a combination of loops or all loops.
• Chimeric libraries of tRNA are also included in the present invention.
• libraries of tRNA synthetases from various organism e.g., microorganisms such as eubacteria or archaebacteria
• libraries that comprise natural diversity see, e.g., U.S. Patent No. 6,238,884 to Short et al; U.S. Patent No. 5,756,316 to Schallenberger et al; U.S. Patent No. 5,783,431 to Petersen et al; U.S. Patent No. 5,824,485 to Thompson et al; U.S. Patent No. 5,958,672 to Short et al
• negatively selecting the library for mutant tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase can include: introducing a toxic marker gene, wherein the toxic marker gene comprises at least one of the selector codons and the library of mutant tRNAs into a plurality of cells from the second organism; and, selecting surviving cells, wherein the surviving cells contain the pool of mutant tRNAs comprising at least one orthogonal tRNA or nonfunctional tRNA.
• the toxic marker gene is a ribonuclease barnase gene, wherein the ribonuclease barnase gene comprises at least one amber codon.
• the ribonuclease barnase gene can include two or more amber codons.
• the surviving cells can be selected, e.g., by using a comparison ratio cell density assay.
• selecting the pool of mutant tRNAs for members that are aminoacylated by an introduced orthogonal RS can include: introducing a positive selection marker gene, wherein the positive selection marker gene comprises a drug resistance gene, e.g., a ⁇ - lactamase gene, comprising at least one of the selector codons, e.g., a ⁇ -lactamase gene comprising at least one amber stop codon, the O-RS, and the pool of mutant tRNAs into a plurality of cells from the second organism; and, selecting surviving cells grown in the presence of a selection agent, e.g., an antibiotic, thereby providing a pool of cells possessing the at least one recombinant tRNA, wherein the recombinant tRNA is aminoacylated by the O-RS and inserts an amino acid into a translation product encoded by the positive marker gene, in response to the at least one selector codons.
• a selection agent e.g., an antibiotic
• the concentration of the selection agent is varied.
• Recombinant O-tRNAs produced by the methods are included in the present invention.
• the stringency of the selection steps e.g., the positive selection step, the negative selection step or both the positive and negative selection steps, in the above described- methods, optionally include varying the selection stringency.
• the stringency of the negative selection can be controlled by introducing different numbers of selector codons into the barnase gene.
• the stringency is varied because the desired activity can be low during early rounds. Thus, less stringent selection criteria are applied in early rounds and more stringent criteria are applied in later rounds of selection.
• selections can be used in the present invention for generating, e.g., O-RS, O-tRNA, and O-tRNA/O-RS pairs.
• the positive selection step, the negative selection step or both the positive and negative selection steps can include using a reporter, wherein the reporter is detected by fluorescence-activated cell sorting (FACS).
• FACS fluorescence-activated cell sorting
• a positive selection can be done first with a positive selection marker, e.g., chloramphenicol acetyltransferase (CAT) gene, where the CAT gene comprises a selector codon, e.g., an amber stop codon, in the CAT gene, which followed by a negative selection screen, that is based on the inability to suppress a selector codon(s), e.g., two or more, at positions within a negative marker, e.g., T7 RNA polymerase gene.
• the positive selection marker and the negative selection marker can be found on the same vector, e.g., plasmid.
• Expression of the negative marker drives expression of the reporter, e.g., green fluorescent protein (GFP).
• the stringency of the selection and screen can be varied, e.g., the intensity of the light need to fluorescence the reporter can be varied.
• a positive selection can be done with a reporter as a positive selection marker, which is screened by FACs, followed by a negative selection screen, that is based on the inability to suppress a selector codon(s), e.g., two or more, at positions within a negative marker, e.g., barnase gene.
• the reporter is displayed on a cell surface, on a phage display or the like.
• Cell-surface display e.g., the OmpA-based cell-surface display system, relies on the expression of a particular epitope, e.g., a poliovirus C3 peptide fused to an outer membrane porin OmpA, on the surface of the Escherichia coli cell.
• the epitope is displayed on the cell surface only when a selector codon in the protein message is suppressed during translation.
• the displayed peptide then contains the amino acid recognized by one of the mutant aminoacyl-tRNA synthetases in the library, and the cell containing the corresponding synthetase gene can be isolated with antibodies raised against peptides containing specific unnatural amino acids.
• the OmpA-based cell-surface display system was developed and optimized by Georgiou et al. as an alternative to phage display. See, Francisco, J. A., Campbell, R., Iverson, B. L. & Georgoiu, G. Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc Natl Acad Sci U S A. 90:10444-8 (1993).
• the selection steps can also be carried out in vitro.
• the selected component e.g., synthetase and/or tRNA, can then be introduced into a cell for use in in vivo inco ⁇ oration of an unnatural amino acid.
• the orthogonal tRNA-RS pair e.g., derived from at least a first, e.g., source organism or at least two source organisms, which can be the same or different, can be used in a variety of host organisms, e.g., a second organism.
• the first and the second organisms of the methods of the present invention can be the same or different.
• the first organism is a prokaryotic organism, e.g., Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like.
• the first organism is a eukaryotic organism, e.g., plants (e.g., complex plants such as monocots, or dicots), algae, protists, fungi (e.g., yeast, etc), animals (e.g., mammals, insects, arthropods, etc.), or the like.
• the second organism is a prokaryotic organism, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like.
• the second organism can be a eukaryotic organism, e.g., plants, fungi, animals, or the like.
• the individual components of a pair can be derived from the same organism or different organisms.
• tRNA can be derived from a prokaryotic organism, e.g., an archaebacterium, such as Methanococcus jannaschii and Halobacterium NRC-1 or a eubacterium, such as Escherichia coli, while the synthetase can be derived from same or another prokaryotic organism, such as, Methanococcus jannaschii, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, P.furiosus, P.
• Eukaryotic sources can also be used, e.g., plants (e.g., complex plants such as monocots, or dicots), algae, protists, fungi (e.g., yeast, etc.), animals (e.g., mammals, insects, arthropods, etc.), or the like.
• plants e.g., complex plants such as monocots, or dicots
• algae e.g., protists, fungi (e.g., yeast, etc.)
• animals e.g., mammals, insects, arthropods, etc.
• Selector codons of the present invention expand the genetic codon framework of protein biosynthetic machinery.
• a selector codon includes, e.g., a unique three base codon, a nonsense codon, such as a stop codon, e.g., an amber codon, or an opal codon, an unnatural codon, at least a four base codon or the like.
• a number of selector codons can be introduced into a desired gene, e.g., one or more, two or more, more than three, etc.
• the 64 genetic codons code for 20 amino acids and 3 stop codons. Because only one stop codon is needed for translational termination, the other two can in principle be used to encode nonproteinogenic amino acids.
• the amber stop codon, UAG has been successfully used in in vitro biosynthetic system and in Xenopus oocytes to direct the inco ⁇ oration of unnatural amino acids.
• UAG is the least used stop codon in Escherichia coli.
• Some Escherichia coli strains contain natural suppressor tRNAs, which recognize UAG and insert a natural amino acid. In addition, these amber suppressor tRNAs have been used in conventional protein mutagenesis.
• the methods involve the use of a selector codon that is a stop codon for the inco ⁇ oration of unnatural amino acids in vivo.
• a selector codon that is a stop codon for the inco ⁇ oration of unnatural amino acids in vivo.
• an O-tRNA is generated that recognizes the stop codon, e.g., UAG, and is aminoacylated by an O-RS with a desired unnatural amino acid.
• This O-tRNA is not recognized by the naturally occurring aminoacyl-tRNA synthetases.
• Conventional site-directed mutagenesis can be used to introduce the stop codon, e.g., TAG, at the site of interest in the protein gene. See, e.g., Sayers, J.R., Schmidt, W. Eckstein, F.
• the inco ⁇ oration of unnatural amino acids in vivo can be done without significant perturbation of the host, e.g., Escherichia coli.
• the suppression efficiency for the UAG codon depends upon the competition between the O-tRNA, e.g., the amber suppressor tRNA, and the release factor 1 (RF1) (which binds to the UAG codon and initiates release of the growing peptide from the ribosome)
• the suppression efficiency can be modulated by, e.g., either increasing the expression level of O-tRNA, e.g., the suppressor tRNA, or using an RF1 deficient strain.
• Unnatural amino acids can also be encoded with rare codons.
• the rare arginine codon, AGG has proven to be efficient for insertion of Ala by a synthetic tRNA acylated with alanine.
• a synthetic tRNA acylated with alanine See, e.g., C. H. Ma, W. Kudlicki, O. W. Odom, G. Kramer and B. Hardesty, Biochemistry, 32:7939 (1993).
• the synthetic tRNA competes with the naturally occurring tRNAArg, which exists as a minor species in Escherichia coli. Some organisms do not use all triplet codons.
• Selector codons also comprise four or more base codons, such as, four, five, six or more base codons. Examples of four base codons include, e.g., AGGA, CUAG, UAGA, CCCU and the like.
• Examples of five base codons include, e.g., AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like.
• mutated O-tRNAs e.g., a special frameshift suppressor tRNAs
• anticodon loops e.g., with at least 8-10 nt anticodon loops
• the four or more base codon is read as single amino acid.
• the anticodon loops can decode, e.g., at least a four-base codon, at least a five-base codon, or at least a six-base codon or more.
• base codons can insert, e.g., one or multiple unnatural amino acids into the same protein.
• four-base codons have been used to inco ⁇ orate unnatural amino acids into proteins using in vitro biosynthetic methods. See, e.g., C. H. Ma, W. Kudlicki, O. W. Odom, G. Kramer and B. Hardesty, Biochemistry, 1993, 32, 7939 (1993); and, T. Hohsaka, D. Kajihara, Y. Ashizuka, H.
• N can be U, A, G, or C
• the quadruplet UAGA can be decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13 to 26% with little decoding in the 0 or -1 frame.
• extended codons based on rare codons or nonsense codons can be used in present invention, which can reduce missense readthrough and frameshift suppression at other unwanted sites.
• a translational bypassing system can also be used to inco ⁇ orate an unnatural amino acid in a desired polypeptide.
• a translational bypassing system a large sequence is inserted into a gene but is not translated into protein. The sequence contains a structure that serves as a cue to induce the ribosome to hop over the sequence and resume translation downstream of the insertion.
• a trans-translation system can be used. This system involves a molecule called tmRNA present in Escherichia coli.
• RNA molecule is structurally related to an alanyl tRNA and is aminoacylated by the alanyl synthetase.
• the difference between tmRNA and tRNA is that the anticodon loop is replaced with a special large sequence.
• This sequence allows the ribosome to resume translation on sequences that have stalled using an open reading frame encoded within the tmRNA as template.
• an orthogonal tmRNA can be generated that is preferentially aminoacylated with an orthogonal synthetase and loaded with an unnatural amino acid. By transcribing a gene using the system, the ribosome stalls at a specific site; the unnatural amino acid is introduced at that site, then translation resumes, using the sequence encoded within the orthogonal tmRNA.
• Selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125.
• Properties of third base pairs include stable and selective base pairing, efficient enzymatic inco ⁇ oration into DNA with high fidelity by a polymerase, and the efficient continued primer extension after synthesis of the nascent unnatural base pair. Descriptions of unnatural base pairs which can be adapted for methods and compositions include, e.g., Hirao, et al., An unnatural base pair for inco ⁇ orating amino acid analogues into protein, Nature Biotechnology, 20:177-182
• the unnatural nucleoside is membrane permeable and is phosphorylated to form the corresponding triphosphate.
• the increased genetic information is stable and not destroyed by cellular enzymes.
• Benner and others took advantage of hydrogen bonding patterns that are different from those in canonical Watson-Crick pairs, the most noteworthy example of which is the iso-C:iso-G pair. See, e.g., C. Switzer, S. E. Moroney and S. A. Benner, J. Am. Chem. Soc, 111:8322 (1989); and, J. A. Piccirilli, T. Krauch, S. E. Moroney and S. A.
• a PICS:PICS self-pair is found to be more stable than natural base pairs, and can be efficiently inco ⁇ orated into DNA by Klenow fragment of Escherichia coli DNA polymerase I (KF). See, e.g., D. L. McMinn, A. K. Ogawa, Y. Q. Wu, J. Q. Liu, P. G. Schultz and F. E. Romesberg, J. Am. Chem. Soc, 121:11586 (1999); and, A. K. Ogawa, Y. Q. Wu, D.
• a 3MN:3MN self-pair can be synthesized by KF with efficiency and selectivity sufficient for biological function. See, e.g., A. K. Ogawa, Y. Q. Wu, M. Berger, P. G. Schultz and F. E. Romesberg, J. Am. Chem. Soc, 122:8803 (2000).
• both bases act as a chain terminator for further replication.
• a mutant DNA polymerase has been recently evolved that can be used to replicate the PICS self pair.
• a 7AI self pair can be replicated.
• an unnatural amino acid refers to any amino acid, modified amino acid, or amino acid analogue other than selenocysteine and the following twenty genetically encoded alpha-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.
• the generic structure of an alpha-amino acid is illustrated by Formula I:
• An unnatural amino acid is typically any structure having Formula I wherein the R group is any substituent other than one used in the twenty natural amino acids. See, e.g., Biochemistry by L. Stryer, 3 rd ed. 1988, Freeman and Company, New York, for structures of the twenty natural amino acids. Note that, the unnatural amino acids of the present invention can be naturally occurring compounds other than the twenty alpha-amino acids above. [144] Because the unnatural amino acids of the invention typically differ from the natural amino acids in side chain only, the unnatural amino acids form amide bonds with other amino acids, e.g., natural or unnatural, in the same manner in which they are formed in naturally occurring proteins. However, the unnatural amino acids have side chain groups that distinguish them from the natural amino acids.
• R in Formula I optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group, or the like or any combination thereof.
• unnatural amino acids of interest include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and or photoisomerizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, e.g., polyethers or long chain hydrocarbons, e.g., greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid
• Z typically comprises OH, NH 2 , SH, NH-R', or S-R';
• X and Y which can be the same or different, typically comprise S or O, and
• R and R' which are optionally the same or different, are typically selected from the same list of constituents for the R group described above for the unnatural amino acids having Formula I as well as hydrogen.
• unnatural amino acids of the invention optionally comprise substitutions in the amino or carboxyl group as illustrated by Formulas II and HI.
• Unnatural amino acids of this type include, but are not limited to, -hydroxy acids, ⁇ -thioacids ⁇ - aminothiocarboxylates, e.g., with side chains corresponding to the common twenty natural amino acids or unnatural side chains.
• substitutions at the ⁇ -carbon optionally include L, D, or ⁇ - ⁇ -disubstituted amino acids such as D-glutamate, D-alanine, D-methyl- O-tyrosine, aminobutyric acid, and the like.
• Other structural alternatives include cyclic amino acids, such as proline analogues as well as 3,4,6,7,8, and 9 membered ring proline analogues, ⁇ and ⁇ amino acids such as substituted ⁇ -alanine and ⁇ -amino butyric acid.
• many unnatural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, and the like.
• Tyrosine analogs include para- substituted tyrosines, ortho-substituted tyrosines, and meta substituted tyrosines, wherein the substituted tyrosine comprises an acetyl group, a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C 6 - C 0 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, a polyether group, a nitro group, or the like.
• multiply substituted aryl rings are also contemplated.
• Glutamine analogs of the invention include, but are not limited to, -hydroxy derivatives, ⁇ -substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives.
• Example phenylalanine analogs include, but are not limited to, meta-substituted phenylalanines, wherein the substituent comprises a hydroxy group, a methoxy group, a methyl group, an allyl group, an acetyl group, or the like.
• unnatural amino acids include, but are not limited to, O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4- allyl-L-tyrosine, a 4-propyl -L-tyrosine, a tri-O-acetyl-GlcNAc ⁇ -serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azido-L-phenylalanine, ap- acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, ap-iodo-phenylalanine, a p-bromophenylalanine, a/7-amino-L
• the unnatural amino acids of the invention are selected or designed to provide additional characteristics unavailable in the twenty natural amino acids.
• unnatural amino acid are optionally designed or selected to modify the biological properties of a protein, e.g., into which they are inco ⁇ orated.
• an unnatural amino acid is optionally modified by inclusion of an unnatural amino acid into a protein: toxicity, biodistribution, solubility, stability, e.g., thermal, hydrolytic, oxidative, resistance to enzymatic degradation, and the like, facility of purification and processing, structural properties, spectroscopic properties, chemical and/or photochemical properties, catalytic activity, redox potential, half-life, ability to react with other molecules, e.g., covalentiy or noncovalently, and the like.
• Chemical Synthesis of Unnatural Amino Aicds [153] Many of the unnatural amino acids provided above are commercially available, e.g., from Sigma (USA) or Aldrich (Milwaukee, WI, USA).
• NBS N-bromosuccinimide
• a meta- substituted methylbenzene compound to give a meta-substituted benzyl bromide, which is then reacted with a malonate compound to give the meta substituted phenylalanine.
• Typical substituents used for the meta position include, but are not limited to, ketones, methoxy groups, alkyls, acetyls, and the like.
• 3-acetyl-phenylalanine is made by reacting NBS with a solution of 3-methylacetophenone. For more details see the examples below. A similar synthesis is used to produce a 3-methoxy phenylalanine.
• the design of unnatural amino acids is biased by known information about the active sites of synthetases, e.g., orthogonal tRNA synthetases used to aminoacylate an orthogonal tRNA.
• synthetases e.g., orthogonal tRNA synthetases used to aminoacylate an orthogonal tRNA.
• orthogonal tRNA synthetases used to aminoacylate an orthogonal tRNA.
• three classes of glutamine analogs are provided, including derivatives substituted at the nitrogen of amide (1), a methyl group at the ⁇ -position (2), and a N-C ⁇ -cyclic derivative (3). Based upon the x-ray crystal structure of E.
• coli GlnRS in which the key binding site residues are homologous to yeast GlnRS, the analogs were designed to complement an array of side chain mutations of residues within a 10 A shell of the side chain of glutamine, e.g., a mutation of the active site Phe233 to a small hydrophobic amino acid might be complemented by increased steric bulk at the C ⁇ position of Gin.
• N-phthaloyl-L-glutamic 1,5-anhydride (compound number 4 in Figure 23) is optionally used to synthesize glutamine analogs with substituents at the nitrogen of the amide. See, e.g., King, F.E. & Kidd, D.A.A.
• the anhydride is typically prepared from glutamic acid by first protection of the amine as the phthalimide followed by refluxing in acetic acid. The anhydride is then opened with a number of amines, resulting in a range of substituents at the amide. Deprotection of the phthaloyl group with hydrazine affords a free amino acid as shown in Figure 23.
• Substitution at the ⁇ -position is typically accomplished via alkylation of glutamic acid. See, e.g., Koskinen, A.M.P. & Rapoport, H. Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino Acid Analogues. J. Org. Chem. 54, 1859-1866. (1989).
• a protected amino acid e.g., as illustrated by compound number 5 in Figure 24 is optionally prepared by first alkylation of the amino moiety with 9-bromo-9- phenylfluorene (PhflBr) (see, e.g., Christie, B.D. & Rapoport, H.
• stearothermophilus TyrRS whose active site is highly homologous to that of the M. jannashii synthetase, residues within a lOA shell of the aromatic side chain of tyrosine were mutated (Y32, G34, L65, Q155, D158, A167, Y32 and D158).
• the library of tyrosine analogs, as shown in Figure 26, has been designed to complement an array of substitutions to these active site amino acids. These include a variety of phenyl substitution patterns, which offer different hydrophobic and hydrogen-bonding properties. Tyrosine analogs are optionally prepared using the general strategy illustrated by Figure 27.
• an enolate of diethyl acetamidomalonate is optionally generated using sodium ethoxide.
• a desired tyrosine analog can then be prepared by adding an appropriate benzyl bromide followed by hydrolysis. Cellular uptake of unnatural amino acids
• Unnatural amino acid uptake is one issue that is typically considered when designing and selecting unnatural amino acids, e.g., for inco ⁇ oration into a protein.
• unnatural amino acids e.g., for inco ⁇ oration into a protein.
• the high charge density of ⁇ -amino acids suggests that these compounds are unlikely to be cell permeable.
• Natural amino acids are taken up into bacteria via a collection of protein-based transport systems displaying varying degrees of amino acid specificity. The present invention therefore provides a rapid screen for assessing which unnatural amino acids, if any, are taken up by cells.
• a variety of unnatural amino acids are optionally screened in minimal media for toxicity to cells.
• Toxicities are typically sorted into five groups: (1) no toxicity, in which no significant change in doubling times occurs; (2) low toxicity, in which doubling times increase by less than about 10%; (3) moderate toxicity, in which doubling times increase by about 10% to about 50%; (4) high toxicity, in which doubling times increase by about 50% to about 100%; and (5) extreme toxicity, in which doubling times increase by more than about 100%.
• no toxicity in which no significant change in doubling times occurs
• low toxicity in which doubling times increase by less than about 10%
• moderate toxicity in which doubling times increase by about 10% to about 50%
• (4) high toxicity in which doubling times increase by about 50% to about 100%
• extreme toxicity in which doubling times increase by more than about 100%.
• toxicity of the amino acids scoring as highly or extremely toxic is typically measured as a function of their concentration to obtain IC50 values.
• amino acids which are very close analogs of natural amino acids or which display reactive functionality demonstrate the highest toxicities.
• the former trend suggests that mechanisms of toxicity for these unnatural amino acids can be inco ⁇ oration into proteins or inhibition of essential enzymes that process natural amino acids.
• toxicity assays are optionally repeated at IC50 levels, e.g., in media supplemented with an excess of a structurally similar natural amino acid.
• the presence of excess natural amino acid typically rescues the ability of the cells to grow in the presence of the toxin, presumably because the natural amino acid effectively outcompetes the toxin for either cellular uptake or for binding to essential enzymes.
• the toxic amino acid is optionally assigned a possible uptake pathway and labeled a "lethal allele" whose complementation is required for cell survival.
• lethal alleles are extremely useful for assaying the ability of cells to uptake nontoxic unnatural amino acids. Complementation of the toxic allele, evidenced by the restoration of cell growth, suggests that the nontoxic amino acid is taken up by the cell, possibly by the same uptake pathway as that assigned to the lethal allele. A lack of complementation is inconclusive. For example studies and conclusions see the examples provided below.
• the method typically requires far less effort than radiolabeling large numbers of compounds and is therefore a more advantageous method for analyzing unnatural; amino acids of interest.
• This general strategy is optionally used to rapidly evaluate the cellular uptake of a wide range of molecules such as nucleic acid base analogs, carbohydrate analogs, or peptide analogs.
• this strategy is optionally used to evaluate the cellular uptake of the unnatural amino aids presented herein.
• the present invention also provides a general method for delivering unnatural amino acids, which is independent of all amino acid uptake pathways.
• This general method relies on uptake via peptide permeases, which transport dipeptides and tripeptides across the cytoplasmic membrane.
• Peptide permeases are not very side-chain specific, and the KD values for their substrates are comparable to KD values of amino acid permeases, e.g., about 0.1 mM to about 10 mM). See, e.g., Nickitenko, A., Trakhanov, S. & Quiocho,
• biosynthetic pathways already exist in cells for the production of amino acids and other compounds. While a biosynthetic method for a particular unnatural amino acid may not exist in nature, e.g., in E. coli, the present invention provide ssuch methods.
• biosynthetic pathways for unnatural amino acids are optionally generated in E. coli by adding new enzymes or modifying existing E. coli pathways. Additional new enzymes are optionally naturally occurring enzymes or artificially evolved enzymes.
• the biosynthesis of p-aminophenylalanine relies on the addition of a combination of known enzymes from other organisms. The genes for these enzymes can be introduced into a cell, e.g., an E.
• coli cell by transforming the cell with a plasmid comprising the genes.
• the genes when expressed in the cell, provide an enzymatic pathway to synthesize the desired compound.
• Examples of the types of enzymes that are optionally added are provided in the examples below. Additional enzymes sequences are found, e.g., in Genbank. Artificially evolved enzymes are also optionally added into a cell in the same manner. In this manner, the cellular machinery and resources of a cell are manipulated to produce unnatural amino acids.
• DesignPathTM developed by Genencor (on the world wide web at genencor.com) is optionally used for metabolic pathway engineering, e.g., to engineer a pathway to create O-methyl-L-trosine in E coli.
• This technology reconstructs existing pathways in host organisms using a combination of new genes, e.g., identified through functional genomics, and molecular evolution and design.
• Diversa Co ⁇ oration (on the world wide web at diversa.com) also provides technology for rapidly screening libraries of genes and gene pathways, e.g., to create new pathways.
• the biosynthesis methods of the present invention do not affect the concentration of other amino acids produced in the cell.
• a pathway used to produce pAF from chorismate produces pAF in the cell while the concentrations of other aromatic amino acids typically produced from chorismate are not substantially affected.
• the unnatural amino acid produced with an engineered biosynthetic pathway of the present invention is produced in a concentration sufficient for efficient protein biosynthesis, e.g., a natural cellular amount, but not to such a degree as to affect the concentration of the other amino acids or exhaust cellular resources.
• Typical concentrations produced in vivo in this manner are about 10 mM to about 0.05 mM.
• a bacterium is transformed with a plasmid comprising the genes used to produce enzymes desired for a specific pathway and a twenty-first amino acid, e.g., pAF, dopa, O-methyl-L-tyrosine, or the like, is generated, in vivo selections are optionally used to further optimize the production of the unnatural amino acid for both ribosomal protein synthesis and cell growth.
• compositions that include proteins with unnatural amino acids
• the invention provides compositions of matter, including proteins with at least one unnatural amino acid.
• the invention also provides compositions of matter that include proteins with at least one unnatural amino acid produced using the compositions and methods of the invention.
• the proteins are processed and modified in a cell dependent manner, e.g., phosphorylated, glycosylated, folded, membrane bound, etc.
• the composition optionally includes at least about 10 micrograms, e.g., at least about 50 micrograms, at least about 100 micrograms, at least about 500 micrograms, at least about 1 milligram, or even at least about 10 milligrams or more of the protein, e.g., an amount that can be achieved with in vivo protein production methods (details on recombinant protein production and purification are provided herein).
• the protein is optionally present in the composition at a concentration of at least about 10 micrograms per liter, at least about 50 micrograms per liter, at least about 100 micrograms per liter, at least about 500 micrograms per liter, at least about 1 milligram per liter, or at least about 10 milligrams per liter of the protein, or more micrograms or protein per liter, e.g., in a cell lysate, pharmaceutical buffer, or other liquid suspension (e.g., in a volume of, e.g., anywhere from about lnl to about 100L).
• the production of large quantities (e.g., greater that that typically possible with other methods, e.g., in vitro translation) of a protein including at least one unnatural amino acid is a feature of the invention and is an advantage over the prior art.
• the production of large quantities (e.g., greater that that typically possible with other methods, e.g., in vitro translation) of a protein including at least one unnatural amino acid is a feature of the invention and is an advantage over the prior art.
• the ability to synthesize large quantities of proteins containing, e.g., heavy atoms facilitates protein structure determination via, e.g., X-ray cystallography.
• the inco ⁇ oration of an unnatural amino acid can be done to, e.g., tailor changes in protein structure and/or function, e.g., to change size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, accessibility of protease target sites, etc. Proteins that include an unnatural amino acid can have enhanced or even entirely new catalytic or physical properties.
• compositions including proteins that include at least one unnatural amino acid are useful for, e.g., novel therapeutics, diagnostics, catalytic enzymes, binding proteins (e.g., antibodies), and e.g., the study of protein structure and function.
• a composition includes at least one protein with at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more unnatural amino acids.
• the unnatural amino acids can be identical or different (e.g., the protein can include two or more different types of unnatural amino acids, or can include two or more different sites having unnatural amino acids, or both).
• any protein that includes an unnatural amino acid and any corresponding coding nucleic acid, e.g., which includes one or more selector codons
• therapeutic and other proteins that can be modified to comprise one or more unnatural include, e.g., Alpha-1 antitrypsin, Angiostatin, Antihemolytic factor, antibodies (further details on antibodies are found below), Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, C-X-C chemokines (e.g., T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractant protein- 1, Monocyte chemoattractant protein-2, Monocyte chemoattractant protein-3, Monocyte inflammatory protein- 1 alpha, Monocyte inflammatory protein-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T6426
• therapeutically relevant properties include serum half- life, shelf half-life, stability, immunogenicity, therapeutic activity, detectability (e.g., by the inclusion of reporter groups (e.g., labels or label binding sites) in the unnatural amino acids), reduction of LD-50 or other side effects, ability to enter the body through the gastric tract (e.g., oral availability), or the like.
• reporter groups e.g., labels or label binding sites
• detectability e.g., by the inclusion of reporter groups (e.g., labels or label binding sites) in the unnatural amino acids
• reduction of LD-50 or other side effects e.g., ability to enter the body through the gastric tract (e.g., oral availability), or the like.
• transcriptional and expression activators include genes and proteins that modulate cell growth, differentiation, regulation, or the like.
• Expression and transcriptional activators are found in prokaryotes, viruses, and eukaryotes, including fungi, plants, and animals, including mammals, providing a wide range of therapeutic targets. It will be appreciated that expression and transcriptional activators regulate transcription by many mechanisms, e.g., by binding to receptors, stimulating a signal transduction cascade, regulating expression of transcription factors, binding to promoters and enhancers, binding to proteins that bind to promoters and enhancers, unwinding DNA, splicing pre-mRNA, polyadenylating RNA, and degrading RNA.
• proteins of the invention include expression activators such as cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products, e.g., interleukins (e.g., IL-1, LL-2, LL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF- ⁇ , TGF- ⁇ , EGF, KGF, SCF/c-Kit, CD40I7CD40, VLA-4/VCAM- 1 , ICAM- 1/LFA- 1 , and hyalurin/CD44; signal transduction molecules and corresponding oncogene products, e.g., Mos, Ras, Raf, and Met; and transcriptional activators and suppressors, e.g., p53, Tat, Fos, Myc, Jun, Myb, Rel, and steroid hormone receptors such as those for
• proteins can also be modified to include one or more unnatural amino acid of the invention.
• the invention can include substituting one or more natural amino acids in one or more vaccine proteins with an unnatural amino acid, e.g., in proteins from infectious fungi, e.g., Aspergillus, Candida species; bacteria, particularly E.
• coli which serves a model for pathogenic bacteria, as well as medically important bacteria such as Staphylococci (e.g., aureus), or Streptococci (e.g., pneumoniae); protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as ( + ) RNA viruses (examples include Poxviruses e.g., vaccinia; Picornaviruses, e.g.
• RNA viruses e.g., Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses
• dsDNA viruses Reoviruses, for example
• RNA to DNA viruses i.e., Retroviruses, e.g., HIV and HTLV
• retroviruses e.g., HIV and HTLV
• certain DNA to RNA viruses such as Hepatitis B.
• a variety of enzymes can also be modified to include one or more unnatural amino acid according to the methods herein, such as amidases, amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases, haloperoxidases, monooxygenases (e.g., p450s), Upases, lignin peroxidases, nitrile hydratases, nitrilases, proteases, phosphatases, subtilisins, transaminase, and nucleases.
• unnatural amino acid such as amidases, amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases,
• Agriculturally related proteins such as insect resistance proteins (e.g., the Cry proteins), starch and lipid production enzymes, plant and insect toxins, toxin-resistance proteins, Mycotoxin detoxification proteins, plant growth enzymes (e.g., Ribulose 1,5- Bisphosphate Carboxylase/Oxygenase, "RUBISCO"), lipoxygenase (LOX), and Phosphoenolpyruvate (PEP) carboxylase are also suitable targets for unnatural amino acid modification.
• insect resistance proteins e.g., the Cry proteins
• starch and lipid production enzymes e.g., plant and insect toxins, toxin-resistance proteins, Mycotoxin detoxification proteins
• plant growth enzymes e.g., Ribulose 1,5- Bisphosphate Carboxylase/Oxygenase, "RUBISCO”
• LOX lipoxygenase
• Phosphoenolpyruvate (PEP) carboxylase are also
• nucleic acid for a protein of interest is mutagenized to include one or more selector codon, providing for insertion of the one or more unatural amino acids.
• the present invention includes any such variant, e.g., mutant, versions of any protein, e.g., including at least one unnatural amino acid.
• the present invention also includes corresponding nucleic acids, i.e., any nucleic acid with one or more selector codon that encodes one or more unnatural amino acid.
• the invention provides compositions that include a Aspll2TAG mutant of chloramphenicol acetylransferase (CAT) produced by the compositions and methods of the invention, where the CAT protein includes at least one unnatural amino acid, e.g., an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, an amino- , isopropyl-, or allyl-containing tyrosine analogue, etc., and the protein is present in the composition at a concentration of at least about 100 micrograms per liter.
• CAT protein includes at least one unnatural amino acid, e.g., an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, an amino- , isopropyl-, or allyl-containing tyrosine analogue, etc.
• the invention provides compositions that include a Tyrl63TAG mutant of mouse dihydrofolate reductase (DHFR) where the DHFR protein includes at least one unnatural amino, e.g., an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, an amino-, isopropyl-, or allyl-containing tyrosine analogue, etc., and the protein is present in the composition at a concentration of at least about 100 micrograms per liter.
• DHFR mouse dihydrofolate reductase
• the present invention provides antibodies to unnatural amino acids and to proteins comprising unnatural amino acids.
• Antibodies to unnatural amino acids and proteins comprising such unnatural amino acids are useful as purification reagents, e.g., for purifying the proteins and unnatural amino acids of the invention.
• the antibodies can be used as indicator reagents to indicate the presence of an unnatural amino acid or protein comprising an unnatural amino acid, e.g., to track the presence or location (e.g., in vivo or in situ) of the unnatural amino acid or protein comprising an unnatural amino acid.
• the unnatural amino acid can itself comprise one or more unnatural amino acids, thereby providing an antibody with one or more property conferred by the one or more unnatural amino acids.
• An antibody of the invention can be a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
• Light chains are classified as either kappa or lambda.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
• a typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer.
• Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
• the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
• the terms variable light chain (VL) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.
• Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
• pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab') 2 , a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
• the F(ab') 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab') 2 dimer into an Fab' monomer.
• the Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W.E.
• Antibodies include single chain antibodies, including single chain Fv (sFv or scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
• sFv or scFv single chain Fv
• Antibodies of the invention can be, e.g., polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments, fragments produced by an Fab expression library, or the like.
• antibodies of the invention are valuable, both as general reagents and as therapeutic reagents in a variety of molecular biological or pharmaceutical processes. Methods of producing polyclonal and monoclonal antibodies are available, and can be applied to making the antibodies of the present invention. A number of basic texts describe standard antibody production processes, including, e.g., Borrebaeck (ed) (1995) Antibody Engineering, 2 nd Edition Freeman and Company, NY (Borrebaeck); McCafferty et al.
• antibody libraries can include repertoires of V genes (e.g., harvested from populations of lymphocytes or assembled in vitro) which are cloned for display of associated heavy and light chain variable domains on the surface of filamentous bacteriophage. Phage are selected by binding to an antigen.
• Soluble antibodies are expressed from phage infected bacteria and the antibody can be improved, e.g., via mutagenesis. See e.g., Balint and Larrick (1993) "Antibody Engineering by Parsimonious Mutagenesis” Gene 137:109-118; Stemmer et al. (1993) "Selection of an Active Single
• Kits for cloning and expression of recombinant antibody phage systems are also known and available, e.g., the "recombinant phage antibody system, mouse ScFv module,” from Amersham-Pharmacia Biotechnology (Uppsala, Sweden).
• Bacteriophage antibody libraries have also been produced for making high affinity human antibodies by chain shuffling (See, e.g., Marks et al. (1992) "By- Passing Immunization: Building High Affinity Human Antibodies by Chain Shuffling” Biotechniques 10:779-782. Indeed, antibodies can typically be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc. (www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio. Synthesis, Inc., Research Genetics (Huntsville, Alabama) and many others.
• humanize antibodies of the invention it is useful to "humanize" antibodies of the invention, e.g., where the antibodies are to be administered therapeutically.
• the use of humanized antibodies tends to reduce the incidence of unwanted immune responses against the therapeutic antibodies (e.g., when the patient is a human).
• the antibody references above describe humanization strategies.
• human antibodies are also a feature of the invention. Human antibodies consist of characteristically human immunoglobulin sequences. Human antibodies can be produced in using a wide variety of methods (see, e.g., Larrick et al, U.S. Pat. No. 5,001,065, for a review). A general approach for producing human antibodies by trioma technology is described by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al, U.S. Pat. No. 4,634,666.
• antibodies are useful reagents for ELISA, western blotting, immunochemistry, affinity chromatograpy methods, SPR, and many other methods.
• the references noted above provide details on how to perform
• antibodies of the invention themselves include unnatural amino acids, providing the antibodies with properties of interest (e.g., improved half-life, stability, toxicity, or the like.
• Antibodies account for nearly 50% of all compounds currently in clinical trials (Wittrup, (1999) "Phage on display” Tibtech 17: 423-424 and antibodies are used ubiquitously as diagnostic reagents. Accordingly, the ability to modify antibodies with unnatural amino acids provides an important tool for modifying these valuable reagents.
• MAbs to the field of diagnostics. Assays range from simple spot tests to more involved methods such as the radio-labeled NR-LU-10 MAb from DuPont Merck Co.
• MAbs are central reagents for ELISA, western blotting, immunochemistry, affinity chromatograpy methods and the like. Any such diagnostic antibody can be modified to include one or more unnatural amino acid, altering, e.g., the specificity or avidity of the Ab for a target, or altering one or more detectable property, e.g., by including a detectable label (e.g., spectrographic, fluorescent, luminescent, etc.) in the unnatural amino acid.
• a detectable label e.g., spectrographic, fluorescent, luminescent, etc.
• antibodies can be tumor-specific MAbs that arrest tumor growth by Targeting tumor cells for destruction by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement- mediated lysis (CML) (these general types of Abs are sometimes referred to as "magic bullets").
• ADCC antibody-dependent cell-mediated cytotoxicity
• CML complement- mediated lysis
• One example is Rituxan, an anti-CD20 MAb for the treatment of Non-Hodgkins lymphoma (Scott (1998) "Rituximab: a new therapeutic monoclonal antibody for non- Hodgkin's lymphoma” Cancer Pract 6: 195-7).
• a second example relates to antibodies which interfere with a critical component of tumor growth.
• Herceptin is an anti-HER-2 monoclonal antibody for treatment of metastatic breast cancer, and provides an example of an antibody with this mechanism of action (Baselga et al. (1998) "Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts [published erratum appears in Cancer Res (1999) 59(8):2020], Cancer Res 58: 2825-31).
• a third example relates to antibodies for delivery of cytotoxic compounds (toxins, radionuclides, etc.) directly to a tumor or other site of interest.
• One application Mab is CYT-356, a 90Y-linked antibody that targets radiation directly to prostate tumor cells (Deb et al. (1996) "Treatment of hormone-refractory prostate cancer with 90Y-CYT-356 monoclonal antibody” Clin Cancer Res 2: 1289-97.
• a fourth application is antibody-directed enzyme prodrug therapy, where an enzyme co-localized to a tumor activates a systemically-administered pro-drug in the tumor vicinity.
• an anti-Ep-CAMl antibody linked to carboxypeptidase A is being developed for treatment of colorectal cancer (Wolfe et al.
• Antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase Al in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors GW1031 and GW1843" Bioconjug Chem 10: 38-48).
• Other Abs e.g., antagonists
• An example is Orthoclone OKT3, an anti-CD3 MAb offered by Johnson and Johnson for reducing acute organ transplant rejection (Strate et al. (1990) "Orthoclone OKT3 as first-line therapy in acute renal allograft rejection" Transplant Proc 22: 219-20.
• Another class of antibody products are agonists.
• Mabs are designed to specifically enhance normal cellular functions for therapeutic benefit.
• Mab-based agonists of acetylcholine receptors for neurotherapy are under development (Xie et al. (1997) "Direct demonstration of MuSK involvement in acetylcholine receptor clustering through identification of agonist ScFv" Nat. Biotechnol. 15: 768-71.
• Any of these antibodies can be modified to include one or more unnatural amino acid to enhance one or more therapeutic property (specificity, avidity, serum-half-life, etc.).
• catalytic antibodies such as Ig sequences that have been engineered to mimic the catalytic abilities of enzymes (Wentworth and Janda (1998) "Catalytic antibodies” Curr Opin Chem Biol 2: 138-44.
• catalytic antibodies can also be modified to include one or more unnatural amino acid to improve one or more property of interest.
• Proteins of the invention e.g., proteins comprising unnatural amino acids, antibodies to proteins comprising unnatural amino acids, etc.
• polypeptides of the invention can be recovered and purified by any of a number of methods well known in the art, including, e.g., ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography, gel electrophoresis and the like.
• Protein refolding steps can be used, as desired, in making correctly folded mature proteins.
• High performance liquid chromatography (HPLC), affinity chromatography or other suitable methods can be employed in final purification steps where high purity is desired.
• antibodies made against unnatural amino acids are used as purification reagents, e.g., for affinity-based purification of proteins comprising one or more unnatural amino acid(s).
• the polypeptides are optionally used e.g., as assay components, therapeutic reagents or as immunogens for antibody production.
• proteins can possess a conformation different from the desired conformations of the relevant polypeptides.
• polypeptides produced by prokaryotic systems often are optimized by exposure to chaotropic agents to achieve proper folding.
• the expressed protein is optionally denatured and then renatured. This is accomplished, e.g., by solubilizing the proteins in a chaotropic agent such as guanidine HC1.
• a chaotropic agent such as guanidine HC1.
• guanidine, urea, DTT, DTE, and/or a chaperonin can be added to a translation product of interest.
• Methods of reducing, denaturing and renaturing proteins are well known to those of skill in the art (see, the references above, and Debinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem.,4: 581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270).
• Debinski, et al. describe the denaturation and reduction of inclusion body proteins in guanidine-DTE.
• the proteins can be refolded in a redox buffer containing, e.g., oxidized glutathione and L-arginine. Refolding reagents can be flowed or otherwise moved into contact with the one or more polypeptide or other expression product, or vice-versa.
• the invention provides for nucleic acid polynucleotide sequences and polypeptide amino acid sequences, e.g., O-tRNAs and O- RSs, and, e.g., compositions and methods comprising said sequences.
• Examples of said sequences, e.g., O-tRNAs and O-RSs are disclosed herein.
• the invention is not limited to those sequences disclosed herein, e.g., the Examples.
• the present invention also provides many unrelated sequences with the functions described herein, e.g., encoding an O-tRNA or an O-RS.
• variants of the disclosed sequences are included in the invention. For example, conservative variations of the disclosed sequences that yield a functionally identical sequence are included in the invention. Variants of the nucleic acid polynucleotide sequences, wherein the variants hybridize to at least one disclosed sequence, are considered to be included in the invention. Unique subsequences of the sequences disclosed herein, as determined by, e.g., standard sequence comparison techniques, are also included in the invention.
• amino acid substitutions i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide
• amino acid substitutions are an implied feature of every nucleic acid sequence which encodes an amino acid.
• conservative amino acid substitutions in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct. Such conservative variations of each disclosed sequence are a feature of the present invention.
• Constant variations of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
• nucleic acid sequences or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
• substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded sequence are "conservatively modified variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.
• “conservative variations” of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 2% or 1%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group.
• substitutions of a small percentage, typically less than 5%, more typically less than 2% or 1%, of the amino acids of the polypeptide sequence with a conservatively selected amino acid of the same conservative substitution group.
• sequences which do not alter the encoded activity of a nucleic acid molecule is a conservative variation of the basic nucleic acid.
• Comparative hybridization can be used to identify nucleic acids of the invention, including conservative variations of nucleic acids of the invention, and this comparative hybridization method is a preferred method of distinguishing nucleic acids of the invention.
• target nucleic acids which hybridize to the nucleic acids represented by SEQ ID NO: 1-34 under high, ultra-high and ultra-ultra high stringency conditions are a feature of the invention. Examples of such nucleic acids include those with one or a few silent or conservative nucleic acid substitutions as compared to a given nucleic acid sequence.
• a test nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least Vi as well to the probe as to the perfectly matched complementary target, i.e., with a signal to noise ratio at lest Vi as high as hybridization of the probe to the target under conditions in which the perfectly matched probe binds to the perfectly matched complementary target with a signal to noise ratio that is at least about 5x-10x as high as that observed for hybridization to any of the unmatched target nucleic acids.
• Nucleic acids "hybridize” when they associate, typically in solution. Nucleic acids hybridize due to a variety of well characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like.
• Higgins 1 and Hames and Higgins (1995) Gene Probes 2 IRL Press at Oxford University Press, Oxford, England (Hames and Higgins 2) provide details on the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides.
• An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
• An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, supra for a description of SSC buffer).
• Stringent hybridization wash conditions in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993), supra, and in Hames and Higgins, 1 and 2. Stringent hybridization and wash conditions can easily be determined empirically for any test nucleic acid.
• the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents such as formalin in the hybridization or wash), until a selected set of criteria are met.
• the hybridization and wash conditions are gradually increased until a probe binds to a perfectly matched complementary target with a signal to noise ratio that is at least 5x as high as that observed for hybridization of the probe to an unmatched target.
• "Very stringent" conditions are selected to be equal to the thermal melting point (T m ) for a particular probe.
• the T m is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe.
• "highly stringent” hybridization and wash conditions are selected to be about 5° C lower than the T m for the specific sequence at a defined ionic strength and pH.
• "Ultra high-stringency” hybridization and wash conditions are those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least lOx as high as that observed for hybridization to any of the unmatched target nucleic acids.
• a target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least V2 that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.
• even higher levels of stringency can be determined by gradually increasing the hybridization and/or wash conditions of the relevant hybridization assay. For example, those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least lOx, 20X, 50X, 100X, or 500X or more as high as that observed for hybridization to any of the unmatched target nucleic acids.
• a target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least Vi that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-ultra-high stringency conditions.
• Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
• the invention provides a nucleic acid which comprises a unique subsequence in a nucleic acid selected from the sequences of O-tRNAs and O-RSs disclosed herein.
• the unique subsequence is unique as compared to a nucleic acid corresponding to any known O-tRNA or O-RS nucleic acid sequence. Alignment can be performed using, e.g., BLAST set to default parameters. Any unique subsequence is useful, e.g., as a probe to identify the nucleic acids of the invention.
• the invention includes a polypeptide which comprises a unique subsequence in a polypeptide selected from the sequences of O-RSs disclosed herein.
• the unique subsequence is unique as compared to a polypeptide corresponding to any of known polypeptide sequence.
• the invention also provides for target nucleic acids which hybridizes under stringent conditions to a unique coding oligonucleotide which encodes a unique subsequence in a polypeptide selected from the sequences of O-RSs wherein the unique subsequence is unique as compared to a polypeptide corresponding to any of the control polypeptides (e.g., parental sequences from which synthetases of the invention were derived, e.g., by mutation). Unique sequences are determined as noted above.
• sequence comparison, identity, and homology refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of skill) or by visual inspection.
• nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
• sequence comparison algorithm or by visual inspection.
• the "substantial identity” exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues, or over the full length of the two sequences to be compared.
• sequence comparison and homology determination typically one sequence acts as a reference sequence to which test sequences are compared.
• test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
• sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
• Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 'I. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Ausubel et al, infra).
• HSPs high scoring sequence pairs
• initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
• the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl Acad. Sci. USA 89:10915).
• the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
• polypeptides of the invention provide a variety of new polypeptide sequences (e.g., comprising unnatural amino acids in the case of proteins synthesized in the translation systems herein, or, e.g., in the case of the novel synthetases herein, novel sequences of standard amino acids), the polypeptides also provide new structural features which can be recognized, e.g., in immunological assays.
• the generation of antisera which specifically bind the polypeptides of the invention, as well as the polypeptides which are bound by such antisera, are a feature of the invention.
• the invention includes synthetase proteins that specifically bind to or that are specifically immunoreactive with an antibody or antisera generated against an immunogen comprising an amino acid sequence selected from one or more of (SEQ ID NO:35-66.
• synthetases such as the wild-type Methanococcus jannaschii (M. jannaschii) tyrosyl synthetase (TyrRS). Where the wild-type Methanococcus jannaschii (M.
• TyrRS tyrosyl synthetase
• the immunoassay uses a polyclonal antiserum which was raised against one or more polypeptide comprising one or more of the sequences corresponding to one or more of SEQ ID NO:35-66) or a substantial subsequence thereof (i.e., at least about 30% of the full length sequence provided).
• the set of potential polypeptide immunogens derived from SEQ ID NO:35-66) are collectively referred to below as "the immunogenic polypeptides.”
• the resulting antisera is optionally selected to have low cross-reactivity against the control synthetase homologues and any such cross- reactivity is removed, e.g., by immunoabsorbtion, with one or more of the control synthetase homologues, prior to use of the polyclonal antiserum in the immunoassay.
• one or more of the immunogenic polypeptides is produced and purified as described herein. For example, recombinant protein can be produced in a recombinant cell.
• mice An inbred strain of mice (used in this assay because results are more reproducible due to the virtual genetic identity of the mice) is immunized with the immunogenic protein(s) in combination with a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a standard description of antibody generation, immunoassay formats and conditions that can be used to determine specific immunoreactivity. Additional references and discussion of antibodies is also found herein and can be applied here to defining polypeptides by immunoreactivity).
• a standard adjuvant such as Freund's adjuvant
• a standard mouse immunization protocol see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a standard description of antibody generation, immunoassay formats and conditions that can be used to determine specific immunoreactivity. Additional references and discussion of antibodies is also
• one or more synthetic or recombinant polypeptide derived from the sequences disclosed herein is conjugated to a carrier protein and used as an immunogen.
• Polyclonal sera are collected and titered against the immunogenic polypeptide in an immunoassay, for example, a solid phase immunoassay with one or more of the immunogenic proteins immobilized on a solid support.
• Polyclonal antisera with a titer of 10 6 or greater are selected, pooled and subtracted with the control synthetase polypeptides to produce subtracted pooled titered polyclonal antisera.
• the subtracted pooled titered polyclonal antisera are tested for cross reactivity against the control homologues in a comparative immunoassay.
• discriminatory binding conditions are determined for the subtracted titered polyclonal antisera which result in at least about a 5-10 fold higher signal to noise ratio for binding of the titered polyclonal antisera to the immunogenic synthetase as compared to binding to the control synthetase homologues. That is, the stringency of the binding reaction is adjusted by the addition of non-specific competitors such as albumin or non-fat dry milk, and/or by adjusting salt conditions, temperature, and/or the like.
• test polypeptide a polypeptide being compared to the immunogenic polypeptides and/ or the control polypeptides
• test polypeptides which show at least a 2-5x higher signal to noise ratio than the control synthetase homologues under discriminatory binding conditions, and at least about a V ⁇ signal to noise ratio as compared to the immunogenic polypeptide(s)
• immunoassays in the competitive binding format are used for detection of a test polypeptide.
• cross-reacting antibodies are removed from the pooled antisera mixture by immunoabsorbtion with the control polypeptides.
• the immunogenic polypeptide(s) are then immobilized to a solid support which is exposed to the subtracted pooled antisera.
• Test proteins are added to the assay to compete for binding to the pooled subtracted antisera.
• test protein(s) The ability of the test protein(s) to compete for binding to the pooled subtracted antisera as compared to the immobilized protein(s) is compared to the ability of the immunogenic polypeptide(s) added to the assay to compete for binding (the immunogenic polypeptides compete effectively with the immobilized immunogenic polypeptides for binding to the pooled antisera).
• the percent cross-reactivity for the test proteins is calculated, using standard calculations.
• the ability of the control proteins to compete for binding to the pooled subtracted antisera is optionally determined as compared to the ability of the immunogenic polypeptide(s) to compete for binding to the antisera. Again, the percent cross-reactivity for the control polypeptides is calculated, using standard calculations.
• the test polypeptides are said to specifically bind the pooled subtracted antisera.
• the immunoabsorbed and pooled antisera can be used in a competitive binding immunoassay as described herein to compare any test polypeptide to the immunogenic and/ or control polypeptide(s).
• the immunogenic, test and control polypeptides are each assayed at a wide range of concentrations and the amount of each polypeptide required to inhibit 50% of the binding of the subtracted antisera to, e.g., an immobilized control, test or immunogenic protein is determined using standard techniques. If the amount of the test polypeptide required for binding in the competitive assay is less than twice the amount of the immunogenic polypeptide that is required, then the test polypeptide is said to specifically bind to an antibody generated to the immunogenic protein, provided the amount is at least about 5- lOx as high as for the control polypeptide.
• the pooled antisera is optionally fully immunosorbed with the immunogenic polypeptide(s) (rather than the control polypeptides) until little or no binding of the resulting immunogenic polypeptide subtracted pooled antisera to the immunogenic polypeptide(s) used in the immunosorbtion is detectable.
• This fully immunosorbed antisera is then tested for reactivity with the test polypeptide. If little or no reactivity is observed (i.e., no more than 2x the signal to noise ratio observed for binding of the fully immunosorbed antisera to the immunogenic polypeptide), then the test polypeptide is specifically bound by the antisera elicited by the immunogenic protein.
• mutagenesis the use of vectors, promoters and many other relevant topics related to, e.g., the generation of genes that include selector codons for production of proteins that include unnatural amino acids, orthogonal tRNAs, orthogonal synthetases, and pairs thereof.
• Various types of mutagenesis are used in the present invention, e.g., to insert selector codons that encode unnatural amino acids in a protein.
• mutagenesis include but are not limited to site-directed, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction- purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like. Mutagenesis, e.g., involving chimeric constructs, are also included in the present invention.
• mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, e.g., sequence, sequence comparisons, physical properties, crystal structure or the like. [236] The above texts and examples found herein describe these procedures. Additional information is found in the following publications and references cited within: Ling et al., Approaches to DNA mutagenesis: an overview, Anal Biochem. 254(2): 157-178 (1997); Dale et al., Oligonucleotide-directed random mutagenesis using the phosphorothioate method, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitro mutagenesis, Ann. Rev. Genet.
• Oligonucleotide-directed mutagenesis a simple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol. 154:329-350 (1987); Taylor et al., The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucl. Acids Res.
• the present invention also relates to host cells and organisms for the in vivo inco ⁇ oration of an unnatural amino acid via orthogonal tRNA/RS pairs.
• Host cells are genetically engineered (e.g., transformed, transduced or transfected) with the vectors of this invention, which can be, for example, a cloning vector or an expression vector.
• the vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide.
• the vectors are introduced into cells and/or microorganisms by standard methods including electroporation (From et al., Proc. Natl. Acad. Sci.
• the engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, screening steps, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic organisms.
• Several well-known methods of introducing target nucleic acids into bacterial cells are available, any of which can be used in the present invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors (discussed further, below), etc.
• Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention.
• the bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook).
• kits are commercially available for the purification of plasmids from bacteria, (see, e.g., EasyPrepTM, FlexiPrepTM, both from Pharmacia Biotech; StrataCleanTM, from Stratagene; and, QIAprepTM from Qiagen).
• the isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect cells or inco ⁇ orated into related vectors to infect organisms.
• Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid.
• the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
• Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, et al, Nature, 328:731 (1987); Schneider, B., et al, Protein Expr. Purifi 6435:10 (1995); Ausubel, Sambrook, Berger (all supra). A catalogue of Bacteria and
• Bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al. (eds) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY.
• nucleic acid and virtually any labeled nucleic acid, whether standard or non-standard
• nucleic acid can be custom or standard ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others.
• compositions [242]
• the proteins, e.g., polypeptides, peptides, etc., of the invention are optionally employed for therapeutic uses, e.g., in combination with a suitable pharmaceutical carrier.
• Such compositions e.g., comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient.
• a carrier or excipient includes, but is not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and/or combinations thereof.
• the formulation is made to suit the mode of administration.
• compositions comprising one or more polypeptide of the invention are optionally tested in one or more appropriate in vitro and/or in vivo animal models of disease, to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art.
• dosages can be initially determined by activity, stability or other suitable measures of unnatural herein to natural amino acid homologues (e.g., comparison of an EPO modified to include one or more unnatural amino acids to a natural amino acid EPO), i.e., in a relevant assay.
• Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells.
• the unnatural amino acid polypeptides of the invention are administered in any suitable manner, optionally with one or more pharmaceutically acceptable carriers. Suitable methods of administering such polypeptides in the context of the present invention to a patient are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective action or reaction than another route.
• compositions of the present invention are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.
• Polypeptide compositions can be administered by a number of routes including, but not limited to: oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means. Unnatural amino acid polypeptide compositions can also be administered via liposomes. Such administration routes and appropriate formulations are generally known to those of skill in the art.
• the unnatural amino acid polypeptide can also be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
• Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non- aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
• the formulations of packaged nucleic acid can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
• parenteral administration and intravenous administration are preferred methods of administration.
• the routes of administration already in use for natural amino acid homologue therapeutics e.g., those typically used for EPO, GCSF, GMCSF, IFNs, interleukins, antibodies, and/or any other pharmaceutically delivered protein
• formulations in current use provide preferred routes of administration and formulation for the unnatural amino acids of the invention.
• the dose administered to a patient, in the context of the present invention is sufficient to effect a beneficial therapeutic response in the patient over time, or, e.g., to inhibit infection by a pathogen, or other appropriate activity, depending on the application.
• the dose is determined by the efficacy of the particular vector, or formulation, and the activity, stability or serum half-life of the unnatural amino acid polypeptide employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
• the size of the dose is also determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, formulation, or the like in a particular patient.
• the physician evaluates circulating plasma levels, formulation toxicities, progression of the disease, and/or where relevant, the production of anti- unnatural amino acid polypeptide antibodies.
• the dose administered, e.g., to a 70 kilogram patient are typically in the range equivalent to dosages of currently-used therapeutic proteins, adjusted for the altered activity or serum half-life of the relevant composition.
• the vectors of this invention can supplement treatment conditions by any known conventional therapy, including antibody administration, vaccine administration, administration of cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogues, biologic response modifiers, and the like.
• formulations of the present invention are administered at a rate determined by the LD-50 of the relevant formulation, and/or observation of any side- effects of the unnatural amino acids at various concentrations, e.g., as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.
• a patient undergoing infusion of a formulation develops fevers, chills, or muscle aches, he/she receives the appropriate dose of aspirin, ibuprofen, acetaminophen or other pain fever controlling drug.
• Patients who experience reactions to the infusion such as fever, muscle aches, and chills are premedicated 30 minutes prior to the future infusions with either aspirin, acetaminophen, or, e.g., diphenhydramine.
• Meperidine is used for more severe chills and muscle aches that do not quickly respond to antipyretics and antihistamines. Cell infusion is slowed or discontinued depending upon the severity of the reaction.
• An orthogonal tRNA/synthetase pair in E. coli can be generated by importing a pair from a different organism, if cross-species aminoacylation is inefficient (Y. Kwok, J. T. Wong, Can. J. Biochem. 58, 213-8 (1980)), and the anticodon loop is not a key determinant of synthetase recognition.
• One such candidate pair is the tyrosyl tRNA/synthetase pair of Methanococcus jannaschii (M. jannaschii), an archaebacterium whose tRNA identity elements differ from those of E.
• coli tRNATyr and whose tyrosyl synthetase (TyrRS) lacks an anticodon loop binding domain (B. A. Steer, P. Schimmel, J. Biol. Chem. 274, 35601-6 (1999)).
• M. jannaschii TyrRS does not have an editing mechanism (H. Jakubowski, E. Goldman, Microbiol. Rev. 56, 412-29 (1992)), and therefore should not proofread an unnatural amino acid ligated to the tRNA.
• mutant suppressor tRNA When the best mutant suppressor tRNA ( mtRNAcu ⁇ ) selected from the library was expressed, cells survived at only 12.4 micrograms/mL ampicillin.
• the mutant suppressor mtRNA ⁇ contained the following nucleotide substitutions: C17A, U17aG, U20C, G37A, and U47G.
• cells with pBLAM only in the absence of any suppressor tRNA survive at 9.7 micrograms/mL ampicillin.
• M. jannaschii TyrRS When the M. jannaschii TyrRS is coexpressed with this mtRNA ⁇ r A , cells survive at 436 micrograms/mL ampicillin.
• the mtRNAju A is a poorer substrate for the endogenous synthetases than the M. jannaschii tRNA ⁇ A and is still aminoacylated efficiently by the M. jannaschii TyrRS.
• residues from a mutant M. jannaschii TyrRS are Tyr 32 (Tyr 34 ), Glu 107 (Asn 123 ), Asp 158 (Asp 176 ), De 159 (Phe 177 ), and Leu 162 (Leu 180 ) with B. stearothermophilus TyrRS residues in parenthesis.
• the M. jannaschii TyrRS gene was expressed under the control of E. coli GlnRS promoter and terminator in plasmid pBK-JYRS, a pBR322 derived plasmid with kanamycin resistance. Residues Tyr32, Glul07, Aspl58, Ilel59 and Leul62 were substituted with Ala by site-directed mutagenesis to afford plasmid pBK-JYA5.
• Oligonucleotides LW157 5'-GGAATTCCATATGGACGAATTTGAAATG-3', LW164 5'-GTATTT TACCACTTGGTTCAAAACCTATMNNAGCAGATTTTTCATCTTTTTTTCATCTTT TTTTAAAAC-3', LW159 5'-TAGGTTTTGAACCAAGTGGTAAAATAC-3', LW165 5 ' -C ATTC AGTGTATA ATCCTTATC AAGCTGGA AMNNACTTCC ATA A ACATATTTTGCCTTTAAC-3', LW161 5'-TCCAGCTTGATAAGGATTATACA CTGAATG-3', LW167 5'-CATCCCTCCAACTGCAACATCAACGCCMNNATA ATGMNNMNNATTAACCTGCATTATTGGATAGATAAC-3', LW163 5'-GCGT TGATGTTGCAGTTGGAGGGATG-3', and LW105 5'-AAACTGCAGTTATAAT
• the ligated vectors were transformed into E. coli DH10B competent cells to yield a library of 1.6 X 10 9 colony forming unit (cfu).
• the TyrRS genes from 40 randomly picked colonies were sequenced to confirm that there was no base bias at the randomized NNK positions and no other unexpected mutations.
• the library was amplified by maxiprep, and supercoiled DNA was used to transform the selection strain pYC-J17.
• a positive selection was then applied that is based on suppression of an amber stop codon at a nonessential position (Aspl 12) in the chloramphenicol acetyltransferase (CAT) gene (M. Pastrnak, T. J. Magliery, P. G. Schultz, Helvetica Chimica Acta. 83, 2277-86 (2000)).
• CAT chloramphenicol acetyltransferase
• Cells were grown in media containing the unnatural amino acid and selected for their survival in the presence of various concentration of chloramphenicol. If a mutant TyrRS charges the orthogonal mtRNA ⁇ A with any amino acid, either natural or unnatural, the cell produces CAT and survives.
• the library was then religated into predigested pBK-JYA5 vector to afford a second generation TyrRS library with a typical size of 8 X 108 to 3 X 109 cfu. Thirty randomly selected members from the library were sequenced. The mutagenic rate introduced by DNA shuffling was 0.35%. This library was transformed into the selection strain for the next round of selection followed by shuffling. The concentration of Cm in the positive selection and in plate set 2 was raised to 80 micrograms/mL for the second round and 120 micrograms/mL for the third round; the concentration of Cm in plate set 1 was unchanged.
• DHFR When the mutant TyrRS (LWJ16) was expressed in the presence of mtRNA ⁇ u A and 1 mM O-methyl-L-tyrosine in liquid GMML growth media, full length DHFR was also produced and could be purified by Ni affinity chromatography with an isolated yield of 2 mg/liter. [268] The yield of purified protein is approximately 26 fold lower in liquid GMML media compare to 2YT rich media. For example, when the mtRNA ⁇ r A and wild type M. jannaschii TyrRS are coexpressed, the yield of DHFR is 67 mg/L in 2YT and 2.6 mg/L in liquid GMML.
• IMS ion trap mass spectrometer
• Aspl58Ala and Leul62Pro mutations create a hydrophobic pocket that allows the methyl group of O-methyl-L-tyrosine to extend further into the substrate-binding cavity.
• Pyrophosphate (PPi) exchange was carried out at 37 °C in a reaction mixture containing 100 mM TrisHCl (pH7.5), 10 mM KF, 5 mM MgC12, 2 mM ATP, 2 mM NaPPi, 0.1 mg/mL bovine serum albumin, approximately 0.01 microCi/mL [32P]NaPPi, and various concentrations of tyrosine or O-methyl-L-tyrosine. Reactions were initiated with the addition of the purified mutant TyrRS (LWJ16), and aliquots were periodically taken and quenched with 0.2 M' NaPPi, 7% perchloric acid, and 2% activated charcoal.
• TyrRS M. jannaschii tyrosyl-tRNA synthetase
• This TyrRS does not aminoacylate any endogenous E. coli tRNAs (Steer, B. A.; Schimmel, P. J. Biol. Chem., 274, 35601-35606 (1999)), but aminoacylates the mtRNAcu A with tyrosine (Wang, L.; Magliery, T. J.; Liu, D. R.; Schultz, P. G. J. Am.
• L-3-(2-naphthyl)-alanine was chosen for this study since it represents a significant structural perturbation from tyrosine and may have novel packing properties.
• jannaschii TyrRS that are within 7 A of the para position of the aryl ring of tyrosine were mutated. To reduce the wild-type synthetase contamination in the following selection, these residues (except Ala 167 ) were first all mutated to alanine. The resulting inactive Ala 5 TyrRS gene was used as a template for polymerase chain reaction (PCR) random mutagenesis with oligonucleotides bearing random mutations at the corresponding sites.
• PCR polymerase chain reaction
• the mutant TyrRS library was first passed through a positive selection based on suppression of an amber stop codon at a nonessential position (Aspl 12) in the chloramphenicol acetyltransferase (CAT) gene.
• CAT chloramphenicol acetyltransferase
• the surviving cells were then grown in the presence of chloramphenicol and the absence of the unnatural amino acid. Those cells that did not survive must encode a mutant TyrRS that charges the mtRNA ⁇ with L-3-(2-naphthyl)-alanine, and were picked from a replica plate supplied with the unnatural amino acid. After three rounds of positive selection followed by a negative screen, four mutant TyrRS 's were characterized using an in vivo assay based on the suppression of the Aspll2TAG codon in the CAT gene.
• TyrRS SS12-TyrRS
• IC 5 o 9 ⁇ g/mL chloramphenicol
• IC 5 o 150 ⁇ g/mL chloramphenicol
• the signal-to-noise ratio of more than 1500 observed in the peptide maps shows a fidelity in the inco ⁇ oration of L-3-(2-naphthyl)-alanine of better than 99.8%.
• the evolved SS12-TyrRS has the following mutations: Tyr32 ⁇ Leu32, Aspl58 ⁇ Prol58, Uel59 ⁇ Alal59, Leul62 ⁇ Glnl62, and Alal67 ⁇ Vall67.
• Corresponding residues from B. stearothermophilus are Tyr (Tyr ), Asp (Asp ), He 159 (Phe 177 ), Leu 162 (Leu 180 ), and Ala 167 (Gin 189 ) with B. stearothermophilus TyrRS residues in parenthesis.
• Example 3 In vivo incorporation of amino-. isopropyl-, or allyl-containing tyrosine analogues
• a FACs based screening system was used to rapidly evolve three highly selective synthetase variants that accept amino-, isopropyl-, or allyl-containing tyrosine analogues.
• the system included a multipu ⁇ ose reporter plasmid used for application of both positive and negative selection pressure and for the facile and quantitative evaluation of synthetase activity.
• a chloramphenicol acetyl transferase (CAT) marker allowed positive selection for activity of the M. jannaschii tyrosyl-tRNA synthetase (TyrRS).
• a T7 polymerase/GFP reporter system allowed assessment of synthetase activity within cells grown in both the presence and absence of an unnatural amino acid. Fluorescence activated cell sorting (FACS) was used to screen against synthetase variants that accept natural amino acids, while visual and fluorimetric analyses were to assess synthetase activity qualitatively and quantitatively, respectively.
• FACS Fluorescence
• the system was based on the synthetase-dependent production of GFPuv, a variant of the green fluorescent protein that has been optimized for expression in E. coli (Crameri, A., Whitehom, E.A., Tate, E. & Stemmer, W.P., Nature Biotechnol. 1996, 14, 315-319).
• This fluorophore is amenable to use in FACS and fluorimetry, as well as visual inspection on plates and in liquid culture.
• the system was designed such that synthetase-dependent suppression of selector, e.g., amber nonsense codons would result in the production of a fluorescence signal.
• T7 RNA polymerase gene was placed under control of the arabinose promoter in order to allow facile optimization of the production of the RNA transcript for amber codon-containing T7 RNA polymerase.
• Optimization of the T7 RNA polymerase/GFPuv reporter system A medium- copy reporter plasmid, pREP, was designed to express amber-containing T7 RNA polymerase variants under control of the arabinose promoter and the GFPuv gene under control of the T7 promoter ( Figure 6a).
• T7 RNA polymerase variants designed to optimize synthetase-dependent fluorescence enhancement (Figure 6b), were inserted into pREP to create plasmids pREP(l-12). All variants contained an N-terminal leader sequence of seven amino acids (MTMITVH) and 1-3 amber stop codons (TAG). Variants 1-3 contained one, two, and three amber stop codons, respectively, substituted for the original methionine at position one (Ml), just downstream of the leader sequence. Variants 4-9 contained an amber codon substituted for D10, R96, Q107, A159, Q169, or Q232, respectively, which were predicted to be located in loop regions of the structure (Jeruzalmi, D.
• Variants 10-12 contained amber stop codons substituted at postions Ml and either Q107, A159, or Q232, respectively. Plasmid constructs were evaluated by fluorimetry and flow cytometry of live cells for fluorescence enhancement using a compatible plasmid containing the orthogonal glutaminyl-tRNA synthetase and Glutamine tRNAcu A from S. cerevisiae.
• Plasmids pREP(l-12) were found to provide varying levels of synthetase-dependent fluorescence enhancement, with the best construct, pREP(10) exhibiting 220-fold greater fluorescence by fluorimetry ( Figure 6c) and ⁇ 400-fold greater median fluorescence by cytometry ( Figure 6d) in cells containing the wild type synthetase versus an inactive mutant. Substitution of a variety of functional groups at positions corresponding to the amber codons within pREP(lO) demonstrate that position 107 within T7 RNA polymerase is highly permissive.
• plasmid pREP(10) was combined with plasmid pYC-J17 (Wang, L, Brock, A., Herberich, B. & Schultz, P.G., Science, 2001, 292, 498-500) to obtain pREP/YC-JYCUA ( Figure 7b). Plasmid pREP/YC-JYCUA was assayed for function with a compatible plasmid expressing a variant of M.
• jannaschii TyrRS (pBK-mJYRS; Wang, L, Brock, A., Herberich, B. & Schultz, P.G., Science, 2001, 292, 498-500) selective for inco ⁇ orating O-Methyl-Tyrosine (OMY).
• OMY O-Methyl-Tyrosine
• jannaschii TyrRS by challenging the enzyme to accept four new functionalities: p-Isopropyl-Phenylalanine (piF), p-Amino-Phenylalanine (pAF), p- Carboxyl-Phenylalanine (pCF), or O-Allyl-Tyrosine (OAT) (Figure 7b).
• piF p-Isopropyl-Phenylalanine
• pAF p-Amino-Phenylalanine
• pCF p- Carboxyl-Phenylalanine
• OFAT O-Allyl-Tyrosine
• a myoglobin gene containing an amber codon in the fourth position was used to assess the production of unnatural amino acid-containing protein.
• the gene was expressed in cells, using the pIF-RS, pAF-RS, or OMY-RS variant, respectively, in either the presence or absence of pJF, pAF, or OAT ( Figure 8d). Protein yields were comparable for all three variants, ranging from 1-2 milligrams of protein per liter of unnatural amino acid- containing cell culture. In contrast, protein production was virtually undetectable in cultures grown in the absence of unnatural amino acid.
• Proteins were analyzed by electrospray mass spectrometry, giving masses of 18457.40 ⁇ 0.81 (18457.28 expected) for the p F-containing protein, 18430.30 ⁇ 0.27 (18430.21 expected) for thepAF- containing protein.
• Activity measurements obtained using the Cm IC 5 o, fluorimetry, and protein expression analyses correlated well, however the activity of the pIF-RS appears to be somewhat underestimated by fluorimetry.
• the disproportionately low fluorimetry measurement for the /?JF-RS variant shows that T7 RNA polymerase may be partially destabilized upon inco ⁇ oration of the pIF analogue, despite the apparent permissivity of the amber positions within the reporter.
• the reporter system described here allows the use of a single multipu ⁇ ose plasmid for both positive selection and negative screening, obviating the need to shuttle plasmids between alternating rounds of positive and negative selection. A total of only three rounds of positive selection and one round of negative screening were required to enable the identification of synthetase variants that selectively accept desired unnatural amino acids. These features allow evolution experiments to be carried out in a matter of days.
• the screening system can be used to readily identify active synthetase variants using agar plates containing unnatural amino acid and to individually assay the amino acid specificity of the variants.
• the T7 RNA polymerase/GFP system can be used to quantitatively compare the activities of synthetase variants.
• the availability of the three OAT-RS clones described here and a different OAT-RS clone derived independently from the same library using a positive/negative selection based on CAT and barnase (Table 2) allows the possibility of comparing the two different evolution systems in terms of the synthetase variants resulting from each ( Figure 9). This analysis reveals that the three clones derived from positive selection and negative screening exhibit slightly lower levels of fluorescence in the presence of OAT, but ⁇ 10-fold lower background levels in the absence of the unnatural amino acid.
• the fluorescence enhancement for cells grown in the presence versus the absence of the unnatural amino acid is thus about 6-fold higher for cells expressing OAT-RS(l) from selection and screening than for cells expressing the OAT-RS clone derived from positive/negative selection using barnase.
• the fluorescence enhancement for cells grown in the presence versus the absence of an unnatural amino acid is expected to represent a lower limit for the fidelity of unnatural amino acid inco ⁇ oration, as competition of unnatural amino acids for being bound by an evolved synthetase variant would reduce binding of natural amino acids.
• high fidelity is clearly desirable, there is likely to be a trade-off between fidelity and overall synthetase activity, which may depend on the desired application.
• [300] Generality of aminoacyl tRNA synthetase evolution. Previous results and those presented here demonstrate that the amino acid side chain binding pocket of the M. jannaschii TyrRS is quite malleable. The enzyme can be evolved to accommodate a variety of functionalities in place .
• enzyme can be evolved to accommodate an amine, isopropyl, or allyl ether functionality at the para position of the tyrosine ring, instead of hydroxyl.
• Plasmid Construction Plasmid pREP ( Figure 6a) was constructed by insertion of a Bam ⁇ U/ApaLl overlap PCR fragment containing the T7 RNA polymerase gene upstream of an rraB transcription termination region, followed by an ApaLVAhdl overlap PCR fragment containing the r ⁇ C gene and ara promoter region from the pB AD/ yc-His A plasmid (Invitrogen; for transcriptional control of the T7 RNA polymerase gene) and the GFPuv gene (Clontech; upstream of the T7 terminator region and downstream of the T7 promoter) between the AhdUBamHJ sites of plasmid pACYC177 (New England Biolabs).
• Plasmids pREP(l-12) were constructed by replacement of an HpaUApaLI fragment of T7 RNA polymerase with overlap PCR fragments containing amber mutations at the positions described.
• Plasmid pREP/YC-JYCUA was constructed by ligation of an AfeUSacU fragment from pREP(10) and an E ⁇ rI(blunted)/S ⁇ cII fragment from pYC-J17 (Wang, L, Brock, A., Herberich, B. & Schultz, P.G, Science, 2001, 292, 498-500). The desired construct was identified following transformation into cells containing plasmid pQ screening for fluorescence.
• Plasmid pQ was constructed by triple ligation of a AaiQJSaH overlap PCR fragment containing the ScQRS downstream of the lac promoter region and upstream of the E. coli QRS termination region, a SaWAval overlap PCR fragment containing the S. cerevisiae tRNA(CUA) Gln downstream of the Ipp promoter region and upstream of an rr ⁇ C termination region, and the Avail Aatll fragment of pBR322 (New England Biolabs). Plasmid pQD was constructed by replacement of pQ fragment between Bam ⁇ . and BglJl with a Bam UBglll fragment of the ScQRS (D291A) mutant.
• Plasmid pBAD/JYAMB-4TAG was constructed by insertion of a PCR fragment of the S4Amber mutant of myoglobin, containing a C-terminal 6His-tag, into the pBAD/YC-
• JYCUA plasmid a hybrid of plasmid pYC-J17 (Wang, L, Brock, A., Herberich, B. &
• a 100-mL GMML culture containing Tet, Kn, 75 ⁇ g/mL Cm, and 1 mMpIF, pAF, pCF, or OAY was inoculated with cells from the initial positive selection (500 ⁇ L) and grown to saturation at 37°C (-24-36 hr).
• a 25-mL GMML culture containing Tet, Kn, and 0.02% arabinose (Ara) was inoculated with cells from the second positive selection (100 ⁇ L, pelleted and resuspended in GMML) and grown to saturation at 37°C (-24 hr).
• Ara- induced cells grown in the absence of unnatural amino acids (1 mL) were pelleted and resuspended in 3 mL of phosphate-buffered saline (PBS).
• Cells were sorted for lack of expression of GFPuv using a BDIS FACVantage TS ⁇ cell sorter with a Coherent Ente ⁇ rise ⁇ ion laser with excitation at 351 nm and emissions detected using a 575/25 nm bandpass filter.
• Collected cells were diluted in at least 10 volumes of LB, containing Tet and Kn, and grown to saturation.
• 100 ⁇ L of cells from the negative screen were pelleted, resuspended in GMML, and used to inoculate
• DH10B cells cotransformed with pBAD/JYAMB-4TAG and the appropriate pBK plasmid were used to inoculate a 100-mL GMML starter culture containing Kn and Tet, which was grown to saturation.
• a 500-mL culture containing Kn, Tet, 0.002% Ara, 5 ⁇ M FeCl 3 , and the desired unnatural amino acid (or none) was inoculated with 50 mL of the starter culture and grown to saturation (-18 hr).
• Cultures were pelleted, sonicated, and the myoglobin protein isolated according to the protocol of the QiaExpressionist (Qiagen) His-tag purification kit. Proteins were analyzed electrophoretically on a 12-20% gradient SDS polyacrylamide gel and by electrospray mass spectrometry.
• Example 4 Creation of an autonomous 21 amino acid bacterium [307] As described above, the common twenty amino acids are conserved across all known organisms. However, an expanded genetic code is provided herein, e.g., for added functionality, structure determination and the like. To determine whether the expanded genetic code is advantageous to a cell, e.g., with a particular unnatural amino acid, an autonomous bacterium that produces and inco ⁇ orates the unnatural amino acid of interest is desirable. The present invention provides such an autonomous twenty-one amino acid organism, and the results can be extended to the production of additional amino acid organisms, e.g., 22 amino acid organisms and the like.
• the present invention combines the above technology with a biosynthetic pathway system to produce an autonomous twenty-one amino acid bacterium.
• the present invention addresses the question of whether such organisms have or can be evolved to have an evolutionary advantage over organisms that use the twenty natural amino acids.
• a completely autonomous bacterium typically comprises a biosynthetic pathway system, e.g., for producing an unnatural amino acid, and a translation system for inco ⁇ orating the unnatural amino acid into one or more proteins in the bacterium.
• the translation system typically comprises an aminoacyl synthetase that uniquely utilizes this unnatural amino acid and no other, and a tRNA that is acylated by that synthetase and no other, and which delivers the unnatural amino acid into proteins in response to a codon that does not encode any other amino acid.
• the biosynthetic pathway system genes, aminoacyl synthetase genes, and tRNA genes are typically positioned on separate plasmids to maximize control of the modified bacteria.
• the unnatural amino acid is biosynthetically produced and inco ⁇ orated into proteins in vivo.
• pAF is optionally selected as a unnatural amino acid for an autonomous cell, e.g., based on its interesting physical properties, e.g., ⁇ donating effects, hydrogen bonding properties, and weak basicity, its lack of toxicity to E. coli, and the fact that it is a known secondary metabolite.
• pAF is optionally synthesized in E. coli from chorismate 2 (a biosynthetic intermediate in the synthesis of aromatic amino acids) using the S. Venezuelae enzymes PapA, PapB, and PapC together with an ⁇ . coli aminotrasferase.
• a plasmid e.g., as provided in Figure 15 A is optionaly used to transform a cell to provide a cell that synthesizes its own supply of pAF invivo.
• An example plasmid for use in the biosynthesis of pAF in vivo is provided by S ⁇ Q. ID. NO.:67.
• S ⁇ Q ID NO.:68 provides the sequences for the individual genes pap ABC that encode the enzymes that are used to carry out the conversion of chorismate to pAF.
• a cell is modified to produce an unnatural amino acid, e.g., pAF, O-methyl- L-tyrosine, a glycoslyated amino acids, L-dopa or the like
• the cell is also typically modified by the addition of a translation system for inco ⁇ orating the unnatural amino acid into one or more proteins within the cell.
• the translation system is typically provided to the cell via a separate plasmid than that by which the cell is modified to contain the biosynthetic pathway system as this allows closer control over the functions of the plasmids in the cell, e.g., regarding the number of copies, promoters, etc.
• the translation machinery typically comprises an orthogonal tRNA/RS pair, e.g., as provided by co-filed patent application "Methods and Compositions for the Production of Orthogonal tRNA-tRNA Synthetase Pairs," by Schultz et al. (Attorney Docket Number 54-000130), filed April 19, 2002.
• an orthogonal tRNA/RS pair for pAF is optionally progenerated using a Methanococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) and mutant tyrosine amber suppressor tRNA ( mtRNA ⁇ ⁇ A ) pair as a starting point.
• TyrRS Methanococcus jannaschii tyrosyl-tRNA synthetase
• mutant tyrosine amber suppressor tRNA mtRNA ⁇ ⁇ A
• pAFRS pAF specific synthetase
• a combination of positive selections and negative screens are optionally used to identify a pAFrs enzyme from a library of TyrRS variants containing random amino acids at five positions, e.g., Tyr32, Glul07, Aspl58, Ilel59, and Leul62.
• a single reporter plasmid is optionally used for both selection and screening, e.g., as described in co-filed patent application "Methods and Compositions for the Production of Orthogonal tRNA-tRNA Synthetase Pairs," by Schultz et al. (Attorney Docket Number 54-000130), filed April 19, 2002.
• the positive selection is typically based on suppression of a TAG codon at a permissive position within the chloramphenicol acetyltransferase (CAT) gene, (see, e.g., Wang, L., et al., Expanding the genetic code of Escherichia coli. Science, 2001, 292: p. 498-500 and Pasternak, M., TJ. Magliery, and P.G. Schultz, A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair for evolving an organism with an expanded genetic code. Helvetica Chemica Acta, 2000 83: p. 2277), e.g., by either pAFor an endogenous amino acid.
• CAT chloramphenicol acetyltransferase
• Cells containing the TyrRS library and reporter plasmid grown in liquid culture containing pAF are typically selected for survival, e.g., in the presence of chloramphenicol (Cm).
• Cm chloramphenicol
• the negative screen based on suppression of two UAG stop codons at permissive positions within the T7 RNA polymerase gene drives the expression of green fluorescent protein (GFP).
• GFP green fluorescent protein
• Positively selected cells grown in the absence of pAF and Cm, are then typically screened, e.g., using fluorescence activated cell sorting (FACS) for the lack of fluorescence.
• FACS fluorescence activated cell sorting
• the reporter plasmid, pREP(2)/YC-JYCUA contains the genes for CAT, T7 RNA polymerase, GFP, and mtRNA ⁇ ⁇ A , and a selectable marker for
• the CAT gene contains a TAG codon substitution at position Dl 12.
• the T7 RNA polymerase gene contains a seven-amino acid N-terminal leader peptide and TAG substitutions at Ml and Q107.
• For the positive selection cells were grown in GMML minimal media containing 35 ⁇ g/ml Kn, 25 ⁇ g/ml Tet, 75 ⁇ g/ml Cm, and lmM pAF (Sigma).
• the negative screen cells were grown in GMML media containing 35 ⁇ g/ml Kn, 25 ⁇ g/ml Tet, and 0.002 % arabinose.
• FACS Fluorescence Activated Cell Sorter
• the excitation wavelength was 351 nm and emission was detected using a 575/25 nm bandpass filter.
• Collected cells were diluted into at least 10 volumes of LB, containing Tet and Kn, and grown to saturation.
• papA, papB, and papC genes were PCR amplified from S. Kunststoffuele (ATCC 10712) genomic DNA. Genes, papABC were assembled by overlap PCR and inserted into a pSClOl derived plasmid, pLASC, and maintained by ampicillin selection. Ribosome binding sites (rbs) were from the 5' UTR of LacZ, malE, and cro and placed prior to papA, papB, and papC, respectively. The papABC genes were placed under control of lac and Ipp promotor to afford two pathway plasmids pLASC-lacPW and pLASC-lppPW.
• DH10B was grown with no plasmids to determine the background suppression level of the reporter plasmid. A sample of each cell growth was diluted to an OD of 1.0 (600 nm) with water and 200 ⁇ L was pelleted.
• DH10B produced 1.0 x 10 4 fluorescent units, while background fluorescence (no pAF added) from the reporter system produced 2.5 xlO 4 fluorescent units.
• the lacPW, lppPW, and 1 mM exogenously added pAF produced 7.9 xlO 4 , 3.0 xlO 6 , and 3.0 xlO 4 fluorescent units, respectively. Induction of the lacPW with IPTG was not feasible due its inhibitory affect on the arabinose promotor in the reporter plasmid, (pREP(2)/YC-JYCUA).
• Aromatic amino acid concentration E. coli DH10B cells harboring the pLASC plasmid and pLASC-lacPW or pLASC-lppPW were grown in GMML minimal media (1% glycerol, 0.3mM leucine) containing 110 ⁇ g/ml ampicillin to saturation. Cells grown with exogenously added pAF contained 1 mM amino acid at the start of the growth. Cells were harvested by centrifugation (100 ml), washed, 1 ml of water and 0.2 ml of toluene was added. Cells were shaken at 37 °C 1 lfor 30 minutes and then separated by centrifugation.
• the aqueous layer was filtered (microcon YM-10) and analyzed by HPLC-MS (Agilent 1100): 5-15 ⁇ L of the aqueous layer seperated on Zorbax SB-C18 column (5 ⁇ m, 4.6X150mm) with a gradient of water 1% TFA /acetonitrile 1% TFA (95:5) to (5:95) over 10 minutes.
• Amino acids were identified by abstracting their MW(+1) from the total ion mass spectrum. The area of the abstracted ion was used to calculate amount of amino acids present in each sample. Cellular concentrations were based on the amount of water in the cell pellet, 70% by mass.
• the papABC genes were expressed from pLASC-lacPW or pLASC-lppPW (13) under the control of the native terminator.
• E. coli DH10B cells harboring plasmid pBAD/JYAMB-4TAG, pBK-TyrRS or pBK-pAFRS , and a pLASC derived plasmid (pLASC, pLASC-lacPW or pLASC-lppPW as indicated) were grown in 0.5 L of minimal media containing 0.002 % arabinose.
• Expression trials with exogenous pAF contained a final concentration of 1 mM pAF (Sigma).
• Example 5 In vivo incorporation of O-methyl-L-tyrosine in an E. coli cell which has been genetically engineered to biosvnthesize the unnatural amino acid
• one aspect of the invention is biosynthetic pathways for unnatural amino acids in E. coli. This is accomplished by e.g., addition to the cell of genes for new enzymes or modification of existing E. coli pathways. In this example, E. coli was genetically engineered to produce the unnatural amino acid O-methyl-L-tyrosine.
• Plant O-methyltransferases are enzymes involved in secondary metabolism, which converts a hydroxyl group into a methoxyl group.
• mutant TyrRS library plasmids A library of plasmids encoding mutant M. jannaschii TryRSs directed at et ⁇ -substituted tyrosine derivatives was constructed, generally following the methods described in Example 1. Briefly, six residues (Tyr 32 , Ala 67 , His 70 , Gin 155 , Asp 158 , Ala 167 ) in the active site of M. jannaschii TyrRS that are within 6.9 A of the met ⁇ -position of the aryl ring of bound tyrosine in the crystal structure of Bacillus stearothermophilus TyrRS were mutated to all 20 amino acids at DNA level using the NNK codon scheme as described in Example 1 above.
• the constructed plasmid library pBK-lib contained around lxlO 9 independent clones.
• Evolution of orthogonal tRNA-synthetase pairs for incorporation of m-acetyl phenylalanine After 3 rounds of positive selection and 2 rounds of negative selection, five candidate clones (SEQ ID NO: 17-21) emerged whose survival in chloramphenicol was dependent on the addition of the unnatural amino acid.
• the IC 50 of chloramphenicol resistance for cells harboring the one of the three mutant TyrRS plasmids is 20 ⁇ g/ml.
• the m- methoxy phenylalanine and m-acetyl phenylalanine synthetases selected above were used to inco ⁇ orate the relevant unnatural amino acids in response to an amber codon in DHFR as previously described in Example 1 above.
• As a negative control cells containing both the orthogonal pair of tRNA-synthetase and amber-mutant vector encoding DHFR were grown in the absence of unnatural amino acids.
• the results of protein expression are shown in Figure 10. These results clearly demonstrated the specificity of the orthogonal pair of tRNA-synthetase to inco ⁇ orate unnatural m-methoxy phenylalanine and m-acetyl phenylalanine.
• the yields of expressed DHFR protein are approximately 0.5 mg/L of culture in both cases.
• the fluorescein hydrazide is cell- permeable and does not lyse cells at 4 °C.
• fluorescein hydrazide is cell- permeable and does not lyse cells at 4 °C.
• fluorescein hydrazide it is possible to label the m-acetyl phenylalanine-inco ⁇ orated DHFR protein intra- cellularly with fluorescein hydrazide.
• Cells expressing the "ketone handle"-inco ⁇ orated DHFR were incubated with fluorescein hydrazide solution. After 36 hours at 4 °C and extensive washes to remove excess fluorescein hydrazide, the labeled DHFR protein was purified and subjected to fluorescence emission tests.
• This invention is useful for, e.g., exploring protein interactions. For example, this invention is useful for defining residues in the protein primary sequence that mediate interaction with different cellular components by varying the position of the crosslinker in the protein. Because a covalent bond is formed between the protein and the molecule it interacts with it is possible to detect weak or transient interactions.
• Two chemical functional groups have gained prominence as crosslinkers, aryl- azides and benophenones since they can be activated at wavelengths above 300 nm ( below which protein damage via photooxidation may be a problem).
• a MjTyrRS library of mutants was generated in which five residues (Tyr 34, Glu 107, Asp 158, He 159, Leu 162) were randomized. These residues were chosen on the basis of the crystal structure of Bacillus Stearothermophilus TyrRS complexed with tyrosyl adenylate (P.Brick, T.N.Bhat & D.M.Blow Journal of Molecular Biology 208, 83 (1989)) in which homologous residues (Tyr34, Asnl23, Aspl76, Phel77, Leul80) are within 6 A of the para position of the aryl ring of bound tyrosine.
• the mutant TyrRS library was passed through a positive selection based on suppression of an amber stop codon at a permissive site (Aspl 12) in the chloramphenicol acetyl transferase (CAT) gene.
• CAT chloramphenicol acetyl transferase
• Cells transformed with thesynthetase library, and the CAT mutant were challenged to grow in the presence of lmM pBpa and chloramphenicol.
• Surviving cells contained synthetases capable of charging the orthogonal mtRNA ⁇ A with either a natural or unnatural amino acid.
• synthetase genes were transferred into cells containing mtRNA u A and a variant of the gene encoding the toxic barnase protein, which contains three amber mutations at permissive sites (Gln2, Asp44,Gly65)(Wang, L., Brock, A., Herberich, B. & Schultz, P. G. Science 292, 498-500(2001)). Growth of these cells in the absence of pBpa selected against synthetases capable of utilizing natural amino acids.
• jannaschii TyrRS is converted to alanine or glycine in five of the six mutant synthetase clones. Aspl58 of the M. jannaschii TyrRS is converted to threonine in five of the six selected mutants, while Ilel59 is converted to serine in four of the six mutants. Serine or proline substitutions dominate at position 107 of M. jannaschii TyrRS; Leul62 is conserved in four of the six mutants. A consensus set of mutations (32:Gly, Ala/ 107:Ser, Pro/ 158:Thr/ 159: Ser/ 162: Leu) emerges from this analysis. [343] In vivo incorporation of pBpa into GST.
• DH10B/ pREP(2)/YC-JYCUA were used to inoculate 0.5 mL of LB supplemented with kanamycin and tetracycline to 30, 20 mg/L. After 20 hours growth (37°C, 300 rpm) cells were diluted 10 4 fold in dri20 and replica spotted on two sets of GMML plates. One set of plates were supplemented with kanamycin and tetracycline at 30 and 20 micrograms/L, respectively, and chloramphenicol at concentrations ranging from 0 micrograms/L to 110 micrograms/L. The second set of plates were identical to the first, except that they were supplemented with 1 mM pBpa .
• Protein Expression Plasmid PYC/SjGSTmut which contains the mutant SjGST gene on an arabinose promoter and rrnB terminator, and mtRNA ⁇ r A on a Ipp promoter and rrnC terminator, and a tetracycline resistance marker was co-transformed with a pBK vector expressing p-BpaRS into DH10B E.coli.
• Cells were amplified in 10 mL of 2x-YT containing kanamycin at 30 micrograms/L and tetracycline at 25 micrograms/L before being washed in PBS and used to inoculate 1 L of liquid GMML with the appropriate antibiotics and pBpa to 1 mM. Protein expression was induced at an ODOOO of 0.6 by the addition of arabinose to 0.2% followed by 5 hours growth. Cells were harvested by centrifugation and protein was purified by virtue of a C-terminal hexa-histidine tag using Ni-NTA affinity chromatography.
• Samples were irradiated at 365 nm using a handheld UV lamp (115V, 60 Hz, 0.2 A; Spectronics, NY, USA), for 1 min or 5 min. Samples were removed from the wells and diluted with SDS loading buffer before resolution of products by SDS-PAGE on a 10-20 % gradient gel. SjGST was transferred to PVDF (Biorad) and probed by western blot using goat anti-GST (Pharmacia) and a secondary mouse anti goat HRP conjugate (Sigma). Signal was developed using Super signal West (Pierce) and visualized by exposure on hyperfilm (Amersham).
• the present invention provides meta substituted phenylalanines as shown in Formula IV:
• Formula IV illustrates the structure of 3-acetyl-phenylalanine and Formula V represents 3-methoxy-phenylalanine.
• Meta-substituted phenylalanines are synthesized in a procedure as outlined in Figure 14.
• NBS N-bromosuccinimide
• Typical substituents used for the meta position include, but are not limited to, ketones, methoxy groups, alkyls, acetyls, and the like. A specific example is provided below.
• NBS N-bromosuccinimide was recrystalized from boiling water prior to usage.
• NBS (1.85 g, 10.5 mmol) was added to a solution of 3-methyl acetophone (1.34 g, 10 mmol).
• ALBN (2', 2'-azobisiosbutyronitrile) (0.043 g, 0.25 mmol) was added to the mixture.
• the reaction mixture was refluxed for 4 hours.
• the completion of reaction was checked by TLC (8:l/hexanes: EtOAc). After aqueous workup, the organic solvent was removed and hexanes was added to give solid. The solid was filtered and washed with hexanes and EtOAc. Then the mixture was recystallized with hexanes.
• Example 10 Synthesis of 4-allyl-L-tyrosine [358]
• the present invention provides 4-allyl-L-tyrosine, whose structure is shown in Formula II: ⁇
• Figure 29 provides a library of unnatural amino acids useful for the following screen. Each amino acid wasscreened at 1 mM in glycerol minimal media for toxicity to cells, e.g., to DH10B harboring pBLAM-YQRS and pACYsupA38.
• Toxicities are sorted into five groups: (1) no toxicity, in which no significant change in doubling times occurs; (2) low toxicity, in which doubling times increase by less than about 10% (seen with the following compounds in Figure 29: S63, S69, S74, S75, S81, S95); (3) moderate toxicity, in which doubling times increase by about 10% to about 50% (seen in the following compounds shown in Figure 29: B, M, P, S12, S14, S22, S41, S45, S49, S52, S62, S64, S65, S71, S91, S93, B10); (4) high toxicity, in which doubling times increase by about 50% to about 100% (seen in the following compounds from Figure 29: C, Q, V, BB, S2, S5, S50, S60, S78, S83, S89, S90); and (5) extreme toxicity, in which doubling times increase by more than about 100% (observed for the following compounds from Figure 29: W, S15, S26, S27, S
• amino acids scoring as highly or extremely toxic are typically measured as a function of their concentration to obtain IC50 values.
• amino acids which are very close analogs of natural amino acids e.g., Q, W, S5, S26, S27, S50, S90, S94
• display reactive functionality e.g., S15, S39, S47
• toxicity assays were repeated at IC50 levels (typically 3 ⁇ M to 500 ⁇ M) in media supplemented with an excess (2 mM) of a structurally similar natural amino acid.
• IC50 levels typically 3 ⁇ M to 500 ⁇ M
• excess natural amino acid rescued the ability of the cells to grow in the presence of the toxin, presumably because the natural amino acid effectively outcompeted the toxin for either cellular uptake or for binding to essential enzymes.
• the toxic amino acid can be assigned a possible uptake pathway and labeled a "lethal allele" whose complementation is required for cell survival.
• Lethal alleles identified in this manner (16 of the toxic unnatural amino acids) span ten possible amino acid uptake groups: alanine, glutamic acid, lysine, leucine, methionine, proline, glutamine, arginine, threonine, and tyrosine.
• coli glutamine and glutamic acid transport pathways may tolerate significant perturbations in amino acid structure, including side chain elongation (X and Z), ketone or methylene placement at the ⁇ -position (B, C, S65), carboxamide replacement with a sulfoxide (S61), a known substrate for a bacterial glutamine transporter or hydrazide (S47), also a known glutamine transporter substrate as well as a variety of hybridization changes at the side chain terminus (S60, S62, K, S84). See, e.g., Jucovic, M. & Hartley, R.W. Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.
• Example 12 Biosynthesis of p-aminophenylalanine [367]
• genes relied on in the pathways leading to chloramphenicol and pristinamycin are optionally used.
• these genes produce pAF as a metabolic intermediate. See, e.g., Yanai, K. and e. al., Streptomyces venezuelae genes pap A , papB, papC, in PCT Int. Appl. 2001, Meiji Seika Kaisha Ltd.: Japan, p.
• a biosynthetic pathway for pAF is shown in Figure 15, Panel B.
• pAF is optionally synthesized in E. coli from chorismate (compound 2 In Figure 15, Panel B), which is a biosynthetic intermediate in the synthesis of aromatic amino acids.
• a cell typically uses a chorismate synthase, a chorismate mutase, a dehydrogenase, e.g., a prephenate dehydrogense, and an amino transferase.
• a chorismate synthase e.g., a chorismate mutase
• a dehydrogenase e.g., a prephenate dehydrogense
• amino transferase e.g., using the S. Venezuelae enzymes PapA, PapB, and PapC together with an E.
• coli aminotransferase e.g., as shown in Figure 15, Panel B, PapA, chorismate is used to produce pAF.
• 4-amino-4-deoxychorismate synthase converts chorismate to 4- amino-4-deoxychorismic acid (compound 3 in Figure 15, Panel B), e.g., using ammonia (from glutamine) in a simple addition-elimination reaction.
• PapB and PapC which are analogous to chorismate mutase and prephenate dehydrogenase, respectively, are used to convert 4-amino-4-deoxychorismic acid to 4-amino-4-deoxyprephenic acid (compound 4 in Figure 15, Panel B) and then to p-aminophenyl-pyruvic acid (compound 5 in Figure 15, panel B).
• a non-specific tyrosine aminotransferase e.g., from E. coli is used to convert p- aminophenyl-pyruvic acid to pAF. See, e.g., Escherichia coli and Salmonella, 2nd ed, ed. F.C. Neidhardt. Vol. 1.
• FIG. 13 illustrates a plasmid for use in the biosynthesis of pAF.
• the plasmid depicted comprises S. Kunststoffuele genes papA, papB, and papC cloned into a pSClOl derived pLASC plasmid, e.g., under control of the lac or Ipp promotor.
• the plasmid is used to transform a cell, e.g., a bacterial cell, such that cell produces the enzymes encoded by the genes.
• the enzymes catalyze one or more reactions designed to produce a desired unnatural amino acid, e.g., pAF.
• a desired unnatural amino acid e.g., pAF.
• proteins PapA, PapB and PapC convert chorismate to p-aminophenyl-pyruvic acid, while an E. coli aromatic aminotransferase completes the biosynthesis to afford pAF.
• the synthesis of pAF from chorismate does not affect the concentration of other amino acids produced in the cell, e.g., other aromatic amino acids typically produced from chorismate.
• p-aminophenylalanine is produced in a concentration sufficient for efficient protein biosynthesis, e.g., a natural cellular amount, but not to such a degree as to affect the concentration of the other aromatic amino acids or exhaust cellular resources.
• Typical concentrations of pAF produced in vivo in this manner are about 10 mM to about 0.05 mM.
• the regulation of the shikimate pathway is modified to account for chorismate consumption in making a fourth aromatic amino acid.
• Example 13 Biosynthesis of Dopa [373]
• hpaBC for a nonspecific aromatic hydroxylase, e.g., from E. coli are cloned into a low copy number vector, e.g., a pSClOl derivative, which is typically placed under control of an Ipp promotor.
• This construct produces dopa (2) from tyrosine (1), in vivo, as shown in Figure 20 while not being toxic to the growing cells. Similar work was done with this gene to ove ⁇ roduce dopa for purification pu ⁇ oses. See, e.g., Jang- Young Lee, Luying Xun,
• O-methyl-L-tyrosine is optionally produced biosynthetically by plant O- methyltransferases are enzymes involved in secondary metabolism, which converts a hydroxyl group into a methoxyl group.
• Two such enzymes were selected: (iso)eugenol O- methyltransferase (JEMT) and caffeic acid O-methyltransferase (COMT). Both of them are from Clarkia breweri.
• IEMT methylates eugenol/isoeugenol, and COMT methylates caffeic acid.
• the substrates of these two enzymes are similar to tyrosine. However, both enzymes have high substrate specificity and methylation regiospecificity.
• the enzymes used to produce O-methyl-L-tyrosine can also be artificially evolved, e.g., to produce a meta substituted methoxy phenylalanine as provided in Formula III.
• the present invention also provides biosynthetic methods for the production of glycosylated amino acids.
• Forming glycosylated amino acids in vivo is optionally performed in a number of ways. For example, transforming a cell with a plasmid comprising a gene for a N-acetyl-galactosaminidase, a transglycosylase, or a hydralase, e.g., serine-glycosyl hydralase, e.g., acting in the reverse direction, provides a cell that produces a glycosylated amino acid.
• the biosynthetic pathway results in a cell that produces and inco ⁇ orates a glycosylated amino acid into one or more proteins within the cell.
• R is optionally an alcohol, an amine, or an N-acetyl amine.
• An example structure is shown by Formula IV: rv
• the approach combines new sets of translational machinery for inco ⁇ oration of unnatural amino acids into proteins with a mutagenized E. coli genomic library placed under selective pressures.
• the genetic, selection approach described above and elsewhere by Schultz and coworkers has, thus far, produced at least about eleven new aminoacyl synthetases that can inco ⁇ orate novel amino acid into proteins efficiently and with high fidelity in response to the TAG codon.
• a pSClOl low copy vector (approximately 5 copies/cell) is optionally used to construct a large insert (7-14 kb) E. coli genomic library by standard methods known to those of skill in the art.
• a 600 member pSClOl based ⁇ . coli genomic library provides complete coverage of the E. coli genome and is also compatible with the aminoacyl synthetase, and tRNA plasmids described above.
• Many mutagens have been studied for there ability to inco ⁇ orate TAG codons into genes. See, e.g., Miller, J.H., A short course in bacterial genetics. 1992, Plainview: Cold Spring Harbor Laboritory Press.
• mutagenesis methods are optionally used since each mutagen is not completely random in its formation of TAG codons.
• TAG codons are typically placed in as many sites as possible.
• Four optional methods include, but are not limited to, UV irradiation, a mutator strain (XL1 red), 4-nitro-quinoline-l -oxide (NQO), and ethylmethane sulfonate (EMS) to mutate the genomic library, and combine them to make one large mutated genomic libraries of >1010 members.
• any of a variety of selective pressures are optionally used for screening that target a range of cellular biology: catalytic functions, protein interactions, carbon sources, multiple response genes, and broad metabolic functions.
• selection pressure based on quinolones is used to target topoisomerase and DNA gyrase.
• 5-fluorouracil is used to target DNA synthesis;
• omeprazole is used to target proton pump inhibitors;
• the use of fatty acids as a sole carbon source and acidic media are used to target a variety of genes related to utilization of carbon and response; and a reductive media is used to target the thiol-redox pathway and disulfide containing proteins.
• Escheria coli and Salmonella cellular and molecular biology ed. F.C. Neidhardt. Vol. 1. 1996, Washington D. C: ASM press; Slonczewski, J.L. and J.W. Foster, pH- regulated genes and survival at extreme pH.
• Escheria coli and Salmonella cellular and molecular biology ed. F.C. Neidhardt. Vol. 1. 1996, Washington D. C: ASM press; and Ritz, D. and J. Beckwith, Annu. Rev. MicrobioL, 2001. 55: p. 21-48.
• the screening of the mutated genomic library produces a set of mutated genomic fragments that confer a growth advantage under a certain selection pressure. These fragments are compared to determine if they are the same found in screens with no unnatural amino acid present by restriction mapping and sequencing. Fragments that produce a growth enhancement from an unnatural amino acid selection this fragment are optionally re-screened by comparing growth rate with each unnatural amino acid and tyrosine suppressing the TAG codon. See, e.g., Figure 21. This re-screening of selected genomic fragments insures that the unnatural amino acid is the factor in conferring a growth advantage.
• the gene(s) that confers an advantage is optionally isolated and identified, e.g., by digestion and subcloning.
• the protein can be studied to identify how the unnatural amino acid is enhancing cellular function.
• the enhanced protein is optionally purified and compared to a natural protein with both in vitro and in vivo studies. Standard enzyme techniques are optionally used to study protein stability, kinetics, and its interaction with other biosynthetic pathway components.
• thermoautotrophicum Sequence Notes tRNA or
• GGTTATCAGA TCCCCCCGAC ACGTTTAATT AATGCTTTCT CCGCCGGAGA
• AAACCAGTAA AACAAAGCAA CTAGAACATG AAATTGAACA CCTGAGACAA
• CTCGCTCCAG TTCGGGCCGG TATCCACCTC GAGAGGTGGC ACTTTTCGGG
• GGCGATCACC GACAGCGGCC TGCCCGTCCT CGGCGTCTGC CTCGGCCACC
• GGCCGAGACC CTCACCGGCC TGGCCGTCCG CGTCCGGCCG AGGCCGACCC

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